Amphiphilic polymers and methods of use thereof

ABSTRACT

The present invention relates to amphiphilic polymers, and micelles and compositions comprising the same, and their use in a variety of biological settings, including imaging, targeting drugs, or a combination thereof for diagnostic and therapeutic purposes.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Ser.No. 60/672,533, filed Apr. 19, 2005, U.S. Provisional Application Ser.No. 60/672,856, filed Apr. 20, 2005 and U.S. Provisional ApplicationSer. No. 60/732,633 filed Nov. 3, 2005, all of which are herebyincorporated by reference in their entirety.

FIELD OF THE INVENTION

This invention provides amphiphilic polymers, processes for producingthe same and methods of use thereof. Polymers of this invention may beused in diagnostics and imaging, as well as treatments of diseases anddisorders including cancer and gene therapy applications.

BACKGROUND OF THE INVENTION

One of the most fundamental limitations to reducing mortality due to anumber of diseases, including cancer, is the fact that current medicalimaging techniques, such as CT and MRI, provide detailed anatomicalsnapshots of the body but fail to provide accurate, basic informationnecessary to manage the patient's disease optimally.

The limitations are manifested in several ways, such as for example incancer: (1) Small primary tumors go undetected. Even under the bestconditions, tumors smaller than 2 mm (roughly 500,000 cells) cannot beseen. (2) Metastatic disease is grossly underdiagnosed, and patientswith negative scans for metastases at initial presentation routinely goon to develop, and die, from metastatic cancer. (3) Treatment responseto therapy is poorly measured. “Measurable disease” is absent aftersurgical excision of many tumors. The standard of care is to blindlytreat with chemotherapy selected by convention using prior retrospectivestudies and to consider this treatment a success or failure only inretrospect (e.g., failure is when a relapse occurs in less than 5years). Residual metastatic disease can expand undetected. Whenmetastatic disease leads to a tumor that is large enough to be detected(stage 4), it is often too late for anything but a modest extension inpatient lifetime with available treatments.

How can conventional imaging be so far off the mark? One reason is thatconventional radiologic approaches produce their images based upon bulkstructural and anatomical features of the tissue. For example, the imagedisplayed in MRI is that of protons in water or fat as modified byrelative concentration and environment. The degree to which, forexample, a tumor can be visualized on conventional CT or MRI is merely afunction of the ability of that tumor to differentially scatter, absorb,or emit radiation as compared to the surrounding tissue and inherentbackground noise. It is not surprising that this signal has littlesensitivity and specificity for the detection of a tumor.

The signal can be enhanced, however, through the use of targeted probes.Supramolecular assemblies that can be made to form nanosphericalstructures for carrying contrast agent, such as liposomes and polymermicelles, offer potential for improving various imaging modalities.Results with such liposomes, however, have essentially beendisappointing.

Moreover, equally frustrating is a lack of versatile delivery systemsfor therapeutics, targeted delivery, and a reliable means of properdosing and tissue distribution of the therapeutic.

SUMMARY OF THE INVENTION

The invention provides, in one embodiment, an amphiphilic polymer,characterized by the structure of the general formula I:

wherein

-   R is a hydroxyl (OH), O-alkyl, O-Acyl, O-Activating group, SH,    S-alkyl, or an acid activating group such as halogen (Cl, Br, I),    O-vinyl, O-allyl, O-aryl, OCOalkyl, OCOaryl, OCH₂CF₃, NH₂, a    fluorochrome, an indole-containing compound, an antibody or antibody    fragment, a peptide, an oligonucleotide, a drug, a ligand for a    biological target, an immunoconjugate, a chemomimetic functional    group, a glycolipid, a labelling agent, an enzyme, a metal ion    chelate, an enzyme cofactor, a cytotoxic compound, a growth factor,    a hormone, a cytokine, a toxin, a prodrug, an antimetabolite, a    microtubule inhibitor, a radioactive material, a targeting moiety;-   R′ is OH, NH₂, SH;-   each R₁ group is, independently, H,

a fluorochrome, an indole-containing compound, an antibody or antibodyfragment, a peptide, an oligonucleotide, a drug, a ligand for abiological target, an immunoconjugate, a chemomimetic functional group,a glycolipid, a labelling agent, an enzyme, a metal ion chelate, anenzyme cofactor, a cytotoxic compound, a growth factor, a hormone, acytokine, a toxin, a prodrug, an antimetabolite, a microtubuleinhibitor, a radioactive material, a perfluorocarbon, aperfluorocarbon-R₄, a perfluorocarbon-OR₄,

-   each R₂ group is, independently, a fluorochrome, an    indole-containing compound, an antibody or antibody fragment, a    peptide, an oligonucleotide, a drug, a ligand for a biological    target, an immunoconjugate, a chemomimetic functional group, a    glycolipid, a labelling agent, an enzyme, a metal ion chelate, an    enzyme cofactor, a cytotoxic compound, a growth factor, a hormone, a    cytokine, a toxin, a prodrug, an antimetabolite, a microtubule    inhibitor, a radioactive material, a perfluorocarbon, a    perfluorocarbon-R₄, a perfluorocarbon-OR₄,

-   each R₃ group is, independently,

a hydrogen, a hydroxyl (OH), O-alkyl, SH, S-alkyl, or an acid activatinggroup such as halogen (Cl, Br, I), O-vinyl, O-allyl, O-aryl, OCOalkyl,OCOaryl, OCH₂CF₃, NH₂, a fluorochrome, an indole-containing compound, anantibody or antibody fragment, a peptide, an oligonucleotide, a drug, aligand for a biological target, an immunoconjugate, a chemomimeticfunctional group, a glycolipid, a labelling agent, an enzyme, a metalion chelate, an enzyme cofactor, a cytotoxic compound, a growth factor,a hormone, a cytokine, a toxin, a prodrug, an antimetabolite, amicrotubule inhibitor, a radioactive material, a targeting moiety;

-   each R₄ group is, independently, an alkyl group, an alkylene group,    a carboxylate group, a carboxylic acid group, an amino group, an    ammonium group, an alkoxyl group, a hydroxyl group or another    nitrogen, oxygen or sulfur-containing group;-   each A group is, independently, O, NH, S, a fluorochrome,

an indole-containing compound, an antibody or antibody fragment, apeptide, an oligonucleotide, a drug, a ligand for a biological target,an immunoconjugate, a chemomimetic functional group, a glycolipid, alabeling agent, an enzyme, a metal ion chelate, an enzyme cofactor, acytotoxic compound, a growth factor, a hormone, a cytokine, a toxin, aprodrug, an antimetabolite, a microtubule inhibitor, a radioactivematerial, a targeting moiety, an acyl group, an aryl group, a linear orbranched alkenyl group, a linear or branched alkyl group, wherein saidalkyl, alkenyl or aryl group is substituted with a perfluorocarbon,perfluorocarbon-R₄, perfluorocarbon-OR₄, perfluorocarbon-OR₄, or

-   n, m, p, p′ and x are integers; and-   q is an integer between 0-10.

In another embodiment, this invention provides a polymer ischaracterized by the structure of the general formula II:

wherein

R′=OH, NH₂, SH;

R=OH, OAlkyl, OAryl, OAcyl, OActivating group;

R₁ and R₃ are H; and

A=O, NH, S.

In another embodiment, this invention provides an amphiphilic polymer,characterized by the structure of the general formula III:

wherein

-   each R group is, independently: a fluorochrome, an indole-containing    compound, an antibody or antibody fragment, a peptide, an    oligonucleotide, a drug, a ligand for a biological target, an    immunoconjugate, a chemomimetic functional group, a glycolipid, a    labelling agent, an enzyme, a metal ion chelate, an enzyme cofactor,    a cytotoxic compound, a growth factor, a hormone, a cytokine, a    toxin, a prodrug, an antimetabolite, a microtubule inhibitor, a    radioactive material, a targeting moiety, an acyl group, an aryl    group, a linear or branched alkenyl group, a linear or branched    alkyl group, wherein said alkyl, alkenyl or aryl group is    substituted with a perfluorocarbon, perfluorocarbon-R₄,    perfluorocarbon-OR₄, or

-   each R′ group is, independently, a hydrogen, a hydroxyl (OH),    O-alkyl, SH, S-alkyl, or an acid activating group such as halogen    (Cl, Br, I), O-vinyl, O-allyl, O-aryl, OCOalkyl, OCOaryl,    OCH₂CF₃NH₂, SH, an acyl group, a fluorochrome, an indole-containing    compound, an antibody or antibody fragment, a peptide, an    oligonucleotide, a drug, a ligand for a biological target, an    immunoconjugate, a chemomimetic functional group, a glycolipid, a    labelling agent, an enzyme, a metal ion chelate, an enzyme cofactor,    a cytotoxic compound, a growth factor, a hormone, a cytokine, a    toxin, a prodrug, an antimetabolite, a microtubule inhibitor, a    radioactive material, a targeting moiety, or

wherein

-   R′″ is a hydroxyl group, an alkoxyl group or a primary or secondary    amino group, O activating group, SH and S-alkyl;-   R₄ is independently an alkyl group, an alkylene group, a carboxylate    group, a carboxylic acid group, and amino group, an ammonium group,    an alkoxyl group, a hydroxyl group or another nitrogen, oxygen or    sulfur-containing group, a halogen;-   A is a fluorochrome, an indole-containing compound, an antibody or    antibody fragment, a peptide, an oligonucleotide, a drug, a ligand    for a biological target, an immunoconjugate, a chemomimetic    functional group, a glycolipid, a labeling agent, an enzyme, a metal    ion chelate, an enzyme cofactor, a cytotoxic compound, a growth    factor, a hormone, a cytokine, a toxin, a prodrug, an    antimetabolite, a microtubule inhibitor, a radioactive material, a    targeting moiety, an acyl group, an aryl group, a linear or branched    alkenyl group, a linear or branched alkyl group, wherein said alkyl,    alkenyl or aryl group is substituted with a perfluorocarbon,    perfluorocarbon-R₄, perfluorocarbon-OR₄, or

-   p and p′ are integers;-   n is at least 1; and-   m is at least 1.

In another embodiment, this invention provides an amphiphilic polymer,characterized by the structure of the general formula IV:

wherein

-   each R group, independently, is a hydroxyl (OH), OCH, CF₃, NH₂, SH,    S, a fluorochrome, an indole-containing compound, an antibody or    antibody fragment, a peptide, an oligonucleotide, a drug, a ligand    for a biological target, an immunoconjugate, a chemomimetic    functional group, a glycolipid, a labelling agent, an enzyme, a    metal ion chelate, an enzyme cofactor, a cytotoxic compound, a    growth factor, a hormone, a cytokine, a toxin, a prodrug, an    antimetabolite, a microtubule inhibitor, a radioactive material, a    targeting moiety, a halogen, an aryl group, a linear or branched    alkenyl group, a linear or branched alkyl group, wherein said alkyl,    alkenyl or aryl group is substituted with a perfluorocarbon,    perfluorocarbon-R₄, perfluorocarbon-OR₄, or

-   R₄ is independently an alkyl group, an alkylene group, a carboxylate    group, a carboxylic acid group, and amino group, an ammonium group,    an alkoxyl group, a hydroxyl group or another nitrogen, oxygen or    sulfur-containing group-   B or B′ is, independently: alkyl, substituted alkyl, aryl,    substituted aryl, OH, NH₂, OR, NHR;-   x=0-6;-   y=0-6;-   p, p′ are integers;-   n is at least 1; and-   m is at least 1.

In another embodiment, this invention provides an amphiphilic polymer,characterized by the structure of the general formula V:

wherein

-   R is a hydroxyl (OH), O-alkyl, SH, S-alkyl, or an acid activating    group such as halogen (Cl, Br, I), O-vinyl, O-allyl, O-aryl,    OCOalkyl, OCOaryl, OCH₂CF₃NH₂, NH, SH, an acyl group, a    fluorochrome, an indole-containing compound, an antibody or antibody    fragment, a peptide, an oligonucleotide, a drug, a ligand for a    biological target, an immunoconjugate, a chemomimetic functional    group, a glycolipid, a labelling agent, an enzyme, a metal ion    chelate, an enzyme cofactor, a cytotoxic compound, a growth factor,    a hormone, a cytokine, a toxin, a prodrug, an antimetabolite, a    microtubule inhibitor, a radioactive material, a targeting moiety,    an aryl group, a linear or branched alkenyl group, a linear or    branched alkyl group, wherein said alkyl, alkenyl or aryl group is    substituted with a perfluorocarbon, perfluorocarbon-R₄,    perfluorocarbon-OR₄, or

-   R₄ is independently an alkyl group, an alkylene group, a carboxylate    group, a carboxylic acid group, and amino group, an ammonium group,    an alkoxyl group, a hydroxyl group or another nitrogen, oxygen or    sulfur-containing group-   A is, independently: O, S or NH-   B or B′ is, independently: alkyl, substituted alkyl, aryl,    substituted aryl, OH, NH₂, OR, NHR;-   n is an integer from 1-10,000-   Each m, independently, is an integer from 1-1,000;-   y or y′ independently, is an integer from 1-10.

In another embodiment, this invention provides an amphiphilic polymer,characterized by the structure of the general formula VI:

wherein

-   R is a hydroxyl (OH), O-alkyl, SH, S-alkyl, or an acid activating    group such as halogen (Cl, Br, I), O-vinyl, O-allyl, O-aryl,    OCOalkyl, OCOaryl, OCH, CF₃NH₂, NH, SH, an acyl group, a    fluorochrome, an indole-containing compound, an antibody or antibody    fragment, a peptide, an oligonucleotide, a drug, a ligand for a    biological target, an immunoconjugate, a chemomimetic functional    group, a glycolipid, a labelling agent, an enzyme, a metal ion    chelate, an enzyme cofactor, a cytotoxic compound, a growth factor,    a hormone, a cytokine, a toxin, a prodrug, an antimetabolite, a    microtubule inhibitor, a radioactive material, a targeting moiety,    an aryl group, a linear or branched alkenyl group, a linear or    branched alkyl group, wherein said alkyl, alkenyl or aryl group is    substituted with a perfluorocarbon, perfluorocarbon-R₄,    perfluorocarbon-OR₄, or

-   R₄ is independently an alkyl group, an alkylene group, a carboxylate    group, a carboxylic acid group, and amino group, an ammonium group,    an alkoxyl group, a hydroxyl group or another nitrogen, oxygen or    sulfur-containing group;-   T, independently is:

-   z is, independently, a halogen, a nitro group, a hydroxy group, an    amino group, an alkyl group, a substituted alkyl group, an aryl    group, a substituted aryl group, wherein said substituted alkyl or    aryl group is substituted with a perfluorocarbon,    perfluorocarbon-R₄, perfluorocarbon-OR₄, or

-   A is, independently O, S, NH;-   p, p′ are integers;-   each x, independently, is an integer from 1-1000; and-   y is an integer from 1-10,000.

In another embodiment, this invention provides an amphiphilic polymer,characterized by the structure of the general formula VII:

wherein

-   R is a hydroxyl (OH), O-alkyl, SH, S-alkyl, or an acid activating    group such as halogen (Cl, Br, I), O-vinyl, O-allyl, O-aryl,    OCOalkyl, OCOaryl, OCH₂CF₃NH₂, NH, SH, an acyl group, a    fluorochrome, an indole-containing compound, an antibody or antibody    fragment, a peptide, an oligonucleotide, a drug, a ligand for a    biological target, an immunoconjugate, a chemomimetic functional    group, a glycolipid, a labelling agent, an enzyme, a metal ion    chelate, an enzyme cofactor, a cytotoxic compound, a growth factor,    a hormone, a cytokine, a toxin, a prodrug, an antimetabolite, a    microtubule inhibitor, a radioactive material, a targeting moiety,    an aryl group, a linear or branched alkenyl group, a linear or    branched alkyl group, wherein said alkyl, alkenyl or aryl group is    substituted with a perfluorocarbon, perfluorocarbon-R₄,    perfluorocarbon-OR₄, or

-   R₄ is independently an alkyl group, an alkylene group, a carboxylate    group, a carboxylic acid group, and amino group, an ammonium group,    an alkoxyl group, a hydroxyl group or another nitrogen, oxygen or    sulfur-containing group;-   T, independently is:

-   z is, independently, H, alkyl, aryl, NH2, NH-alkyl, NH-acyl,    NH-aryl, OH, O-acyl, O-alkyl, O-aryl, a halogen, a nitro group, a    hydroxy group, a substituted alkyl group, a substituted aryl group,    wherein said substituted alkyl or aryl group is substituted with a    perfluorocarbon, perfluorocarbon-OR₄, or

-   A is, independently O, S, NH;-   p, p′ are integers;-   each y, independently, is an integer from 1-1000; and-   n is an integer from 1-10,000.

In another embodiment, this invention provides a composition or amicelle comprising a polymer of this invention.

In another embodiment, this invention provides a process for producingan amphiphilic polymer comprising perfluorocarbons, the processcomprising the steps of:

-   -   contacting a dialkyl 5-hydroxy-isophthalate, a dialkyl        5-alkoxy-isophthalate, a dialkyl 5-amino-isophthalate, any        derivative thereof or any combination thereof with a        polyethylene glycol to form an amphiphilic copolymer; and    -   linking a perfluorocarbons to said amphiphilic copolymer,        thereby being a process for producing amphiphilic polymers        comprising perfluorocarbons.

In another embodiment, this invention provides a method of imaging acell, the method comprising the steps of contacting a cell with anamphiphilic polymer of this invention and imaging said cell, wherebysaid polymer enables the imaging of said cell.

In another embodiment, this invention provides a method of targeteddelivery of at least one agent in a subject comprising the steps ofadministering to said subject an amphiphilic polymer of this invention,wherein said polymer comprises said agent and a targeting agent.

In another embodiment, this invention provides a method for detectingneoplastic cells in a subject, comprising contacting a cell in, or acell derived from said subject with an effective tumor-detecting amountof an amphiphilic polymer of this invention, wherein said polymercomprises a targeting moiety specific for neoplastic cells; anddetecting any of said polymer associated with neoplastic cells presentin said subject.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a synthetic scheme for the preparation of a basiccopolymer structure.

FIGS. 2 a and 2 b depict schemes for the formation of self-assemblingalternating copolymer micelles.

FIG. 3 depicts micelle nanoparticles with perfluorocarbon side chainsand cargo and ¹⁹F Spectra from perfluorocarbon encapsulated1,1,2,2-tetrahydro perfluorodecanol particles.

FIG. 4 shows cellular uptake of the particles. Cryo transmissionelectron microscopy (FIG. 4A) and confocal microscopy (FIGS. 4D and 4E)were used to qualitatively evaluate cellular uptake. Cellular uptake wasalso evaluated quantitatively (FIGS. 4B, 4C and 4F). INS-1 cells wereincubated at 37° C., with the compound (1 mg/mL), and the uptake wasmeasured.

FIG. 5 shows cellular cytotoxicity, following exposure of the cells tosome polymers of the invention.

FIG. 6 describes the kinetics of cellular uptake and intracellularlocalization of some polymers of the invention.

FIG. 7 depicts a crosslinked iron oxide (CLIO)-EPPT multi-modal imagingprobe. (A) The core protein of the MUC-1 tumor antigen. Theimmunodominant region of the tandem repeat is recognized by the EPPT1peptide derived from an ASM2 monoclonal antibody (45). (B) Synthesis(left) and scheme of the probe (right). (C) The absorption spectrum ofCLIO-EPPT showed the presence of three peaks corresponding to FITC,Cy5.5, and iron oxide nanoparticles. (D) Cell binding assay: cellsexpressing underglycosylated mucin-1 accumulate significantly moreCLIO-EPPT (P<0.05) than uMUC-1-negative tumor or normal cells. (E)Fluorescence-activated cell sorting analysis of the set ofunderglycosylated mucin-1 antigen (uMUC-1)-positive tumor cell lines(BT-20, CAPAN-2, ChaGo-K-1, HT-29, LS174T) showed a shift influorescence in the FL1 and FL4 channels and no shift in the controluMUC-1-negative cell line U87. Fluorescence microscopy showedcolocalization of the FITC and Cy5.5 signal within the set of the samecell lines after incubation with the CLIO-EPPT probe. Left, overlay ofthe bright field and FITC channel; middle, overlay of the bright fieldand Cy5.5 channel; right, overlay of the FITC and Cy5.5 channels. Notethat no fluorescence was observed in FITC or Cy5.5 channels in the U87cell line. Magnification bars=10 μm.

FIG. 8 demonstrates results of imaging of the animals bearingunderglycosylated mucin-1 antigen (uMUC-1)-negative (U87) anduMUC-1-positive (LS174T) tumors. (A) Transverse (top) and coronal(bottom) images showed a significant (52%; P<0.0001) decrease in signalintensity in uMUC-1-positive tumors 24 h after administration of theCLIO-EPPT probe. (B) White light (left), near-infrared fluorescent(NIRF) (middle) images, and a color-coded map (right) of mice bearingbilateral underglycosylated mucin-1 antigen uMUC-1-negative (U87) anduMUC-1-positive (LS174T) tumors. NIRF imaging was performed immediatelyafter the MRI session. (C) White light (top) and NIRF (bottom) images ofLS174T- and U87-excised tumors and muscle tissue. uMUC-1-positive LStumor produced a strong NIRF signal. (D) Dual channel fluorescencemicroscopy of the frozen LS174T tumor section. Green channelfluorescence from the FITC-labeled EPPT peptide (left) colocalized withCy5.5 fluorescence derived from Cy5.5-labeled cross-linked iron oxides(middle). The combination image shows colocalization of two signals(right). Magnification bar=10 μm.

FIG. 9 schematically depicts a first stage of synthesis of theamphiphilic polymer I, accomplished via enzymatic polymerization.

FIG. 10 depicts some embodiments of the alternations for perfluorocarbonside chains that may be synthesized or used according to this invention.

FIG. 11 depicts structures of the polymers with various substituents.Structure of nanospheres and positions available for iodination (11 a).A scheme for the fluorescent labeling of perfluorinated side chains (11b).

FIG. 12 schematically depicts some embodiments of polymer structureswhich may be prepared according to the methods of this invention, whichmay find application as probes for multi-modal imaging.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides, in one embodiment, an amphiphilic polymer,characterized by the structure of the general formula I:

wherein

-   R is a hydroxyl (OH), O-alkyl, O-Acyl, O-Activating group, SH,    S-alkyl, or an acid activating group such as halogen (Cl, Br, I),    O-vinyl, O-allyl, O-aryl, OCOalkyl, OCOaryl, OCH₂CF₃, NH₂, a    fluorochrome, an indole-containing compound, an antibody or antibody    fragment, a peptide, an oligonucleotide, a drug, a ligand for a    biological target, an immunoconjugate, a chemomimetic functional    group, a glycolipid, a labelling agent, an enzyme, a metal ion    chelate, an enzyme cofactor, a cytotoxic compound, a growth factor,    a hormone, a cytokine, a toxin, a prodrug, an antimetabolite, a    microtubule inhibitor, a radioactive material, a targeting moiety;-   R′ is OH, NH₂, SH, OR″, NHR″, SR″;-   Where W′ is a fluorochrome, an indole-containing compound, an    antibody or antibody fragment, a peptide, an oligonucleotide, a    drug, a ligand for a biological target, an immunoconjugate, a    chemomimetic functional group, a glycolipid, a labelling agent, an    enzyme, a metal ion chelate, an enzyme cofactor, a cytotoxic    compound, a growth factor, a hormone, a cytokine, a toxin, a    prodrug, an antimetabolite, a microtubule inhibitor, a radioactive    material, a targeting moiety;-   each R₁ group is, independently, H,

a fluorochrome, an indole-containing compound, an antibody or antibodyfragment, a peptide, an oligonucleotide, a drug, a ligand for abiological target, an immunoconjugate, a chemomimetic functional group,a glycolipid, a labelling agent, an enzyme, a metal ion chelate, anenzyme cofactor, a cytotoxic compound, a growth factor, a hormone, acytokine, a toxin, a prodrug, an antimetabolite, a microtubuleinhibitor, a radioactive material, a perfluorocarbon,

-   a perfluorocarbon-R₄, a perfluorocarbon-OR₄,

OH, NH2, NH, S, SH, O-alkyl;

-   each R₂ group is, independently, a fluorochrome, an    indole-containing compound, an antibody or antibody fragment, a    peptide, an oligonucleotide, a drug, a ligand for a biological    target, an immunoconjugate, a chemomimetic functional group, a    glycolipid, a labelling agent, an enzyme, a metal ion chelate, an    enzyme cofactor, a cytotoxic compound, a growth factor, a hormone, a    cytokine, a toxin, a prodrug, an antimetabolite, a microtubule    inhibitor, a radioactive material, a perfluorocarbon, a    perfluorocarbon-R₄, a perfluorocarbon-OR₄,

-   each R₃ group is, independently,

a hydrogen, a hydroxyl (OH), O-alkyl, SH, S-alkyl, or an acid activatinggroup such as halogen (Cl, Br, I), O-vinyl, O-allyl, O-aryl, OCOalkyl,OCOaryl, OCH₂CF₃, NH₂, a fluorochrome, an indole-containing compound, anantibody or antibody fragment, a peptide, an oligonucleotide, a drug, aligand for a biological target, an immunoconjugate, a chemomimeticfunctional group, a glycolipid, a labelling agent, an enzyme, a metalion chelate, an enzyme cofactor, a cytotoxic compound, a growth factor,a hormone, a cytokine, a toxin, a prodrug, an antimetabolite, amicrotubule inhibitor, a radioactive material, a targeting moiety;

-   each R₄ group is, independently, an alkyl group, an alkylene group,    a carboxylate group, a carboxylic acid group, an amino group, an    ammonium group, an alkoxyl group, a hydroxyl group or another    nitrogen, oxygen or sulfur-containing group, a halogen;-   each A group is, independently, O, NH, S, a fluorochrome,

an indole-containing compound, an antibody or antibody fragment, apeptide; an oligonucleotide, a drug, a ligand for a biological target,an immunoconjugate, a chemomimetic functional group, a glycolipid, alabeling agent, an enzyme, a metal ion chelate, an enzyme cofactor, acytotoxic compound, a growth factor, a hormone, a cytokine, a toxin, aprodrug, an antimetabolite, a microtubule inhibitor, a radioactivematerial, a targeting moiety, an acyl group, an aryl group, a linear orbranched alkenyl group, a linear or branched alkyl group, wherein saidalkyl, alkenyl or aryl group is substituted with a perfluorocarbon,perfluorocarbon-R₄, perfluorocarbon-OR₄, perfluorocarbon-OR₄, or

-   n, m, p, p′ and x are integers; and-   q is an integer between 0-10.

In another embodiment, this invention provides a polymer ischaracterized by the structure of the general formula II:

wherein

R′=OH, NH₂, SH, OAlkyl, OAryl, OAcyl, OActivating group;

R=OH, NH₂, SH, OAlkyl, OAryl, OAcyl, OActivating group;

R₁=H;

R₃=H, a hydroxyl (OH), O-alkyl, SH, S-alkyl, or an acid activating groupsuch as halogen (Cl, Br, I), O-vinyl, O-allyl, O-aryl, OCOalkyl,OCOaryl, OCH₂CF₃, NH₂, (a fluorochrome, an indole-containing compound,an antibody or antibody fragment, a peptide, an oligonucleotide, a drug,a ligand for a biological target, an immunoconjugate, a chemomimeticfunctional group, a glycolipid, a labelling agent, an enzyme, a metalion chelate, an enzyme cofactor, a cytotoxic compound, a growth factor,a hormone, a cytokine, a toxin, a prodrug, an antimetabolite, amicrotubule inhibitor, a radioactive material, a targeting moiety); and

A=O, NH, S.

In another embodiment, this invention provides an amphiphilic polymer,characterized by the structure of the general formula III:

wherein

-   each R group is, independently: a fluorochrome, an indole-containing    compound, an antibody or antibody fragment, a peptide, an    oligonucleotide, a drug, a ligand for a biological target, an    immunoconjugate, a chemomimetic functional group, a glycolipid, a    labelling agent, an enzyme, a metal ion chelate, an enzyme cofactor,    a cytotoxic compound, a growth factor, a hormone, a cytokine, a    toxin, a prodrug, an antimetabolite, a microtubule inhibitor, a    radioactive material, a targeting moiety, an acyl group, an aryl    group, a linear or branched alkenyl group, a linear or branched    alkyl group, wherein said alkyl, alkenyl or aryl group is    substituted with a perfluorocarbon, perfluorocarbon-R₄,    perfluorocarbon-OR₄, or

-   each R′ group is, independently, a hydrogen, a hydroxyl (OH),    O-alkyl, SH, S-alkyl, or an acid activating group such as halogen    (Cl, Br, I), O-vinyl, O-allyl, O-aryl, OCOalkyl, OCOaryl,    OCH₂CF₃NH₂, SH, an acyl group, a fluorochrome, an indole-containing    compound, an antibody or antibody fragment, a peptide, an    oligonucleotide, a drug, a ligand for a biological target, an    immunoconjugate, a chemomimetic functional group, a glycolipid, a    labelling agent, an enzyme, a metal ion chelate, an enzyme cofactor,    a cytotoxic compound, a growth factor, a hormone, a cytokine, a    toxin, a prodrug, an antimetabolite, a microtubule inhibitor, a    radioactive material, a targeting moiety, or OR“, NHR”, SR″-   wherein R″ is a fluorochrome, an indole-containing compound, an    antibody or antibody fragment, a peptide, an oligonucleotide, a    drug, a ligand for a biological target, an immunoconjugate, a    chemomimetic functional group, a glycolipid, a labelling agent, an    enzyme, a metal ion chelate, an enzyme cofactor, a cytotoxic    compound, a growth factor, a hormone, a cytokine, a toxin, a    prodrug, an antimetabolite, a microtubule inhibitor, a radioactive    material, a targeting moiety;-   wherein R′″ is a hydroxyl group, an alkoxyl group or a primary or    secondary amino group, O activating group, SH and S-alkyl;-   R₂ is, independently, a fluorochrome, an indole-containing compound,    an antibody or antibody fragment, a peptide, an oligonucleotide, a    drug, a ligand for a biological target, an immunoconjugate, a    chemomimetic functional group, a glycolipid, a labelling agent, an    enzyme, a metal ion chelate, an enzyme cofactor, a cytotoxic    compound, a growth factor, a hormone, a cytokine, a toxin, a    prodrug, an antimetabolite, a microtubule inhibitor, a radioactive    material, a perfluorocarbon, a perfluorocarbon-R₄, a    perfluorocarbon-OR₄,

-   R₄ is independently an alkyl group, an alkylene group, a carboxylate    group, a carboxylic acid group, and amino group, an ammonium group,    an alkoxyl group, a hydroxyl group or another nitrogen, oxygen or    sulfur-containing group, a halogen;-   A is a fluorochrome, an indole-containing compound, an antibody or    antibody fragment, a peptide, an oligonucleotide, a drug, a ligand    for a biological target, an immunoconjugate, a chemomimetic    functional group, a glycolipid, a labeling agent, an enzyme, a metal    ion chelate, an enzyme cofactor, a cytotoxic compound, a growth    factor, a hormone, a cytokine, a toxin, a prodrug, an    antimetabolite, a microtubule inhibitor, a radioactive material, a    targeting moiety, an acyl group, an aryl group, a linear or branched    alkenyl group, a linear or branched alkyl group, wherein said alkyl,    alkenyl or aryl group is substituted with a perfluorocarbon,    perfluorocarbon-R₄, perfluorocarbon-OR₄, or

-   p and p′ are integers;-   n is at least 1; and-   m is at least 1.

In another embodiment, this invention provides an amphiphilic polymer,characterized by the structure of the general formula IV:

wherein

-   each R group, independently, is a hydroxyl (OH), OCH₂CF₃, NH₂, SH,    S, a fluorochrome, an indole-containing compound, an antibody or    antibody fragment, a peptide, an oligonucleotide, a drug, a ligand    for a biological target, an immunoconjugate, a chemomimetic    functional group, a glycolipid, a labelling agent, an enzyme, a    metal ion chelate, an enzyme cofactor, a cytotoxic compound, a    growth factor, a hormone, a cytokine, a toxin, a prodrug, an    antimetabolite, a microtubule inhibitor, a radioactive material, a    targeting moiety, a halogen, an aryl group, a linear or branched    alkenyl group, a linear or branched alkyl group, wherein said alkyl,    alkenyl or aryl group is substituted with a perfluorocarbon,    perfluorocarbon-R₄, perfluorocarbon-OR₄, or

-   R₄ is independently an alkyl group, an alkylene group, a carboxylate    group, a carboxylic acid group, and amino group, an ammonium group,    an alkoxyl group, a hydroxyl group, a halogen or another nitrogen,    oxygen or sulfur-containing group-   B or B′ is, independently: alkyl, substituted alkyl, aryl,    substituted aryl, OH, NH₂, OR₁, NHR₁; OCOR₁, NHCOR₁-   Where R₁ is alkyl, substituted alkyl, aryl, substituted aryl,    wherein the said alkyl or aryl group is either perfluorinated or    substituted with perfluorinated compound.-   x=0-10;-   y=0-10;-   p, p′ are integers;-   n is at least 1; and-   m is at least 1.

In another embodiment, this invention provides an amphiphilic polymer,characterized by the structure of the general formula V:

wherein

-   R is a hydroxyl (OH), O-alkyl, SH, S-alkyl, or an acid activating    group such as halogen (Cl, Br, I), O-vinyl, O-allyl, O-aryl,    OCOalkyl, OCOaryl, OCH₂CF₃NH₂, NH, SH, an acyl group, a    fluorochrome, an indole-containing compound, an antibody or antibody    fragment, a peptide, an oligonucleotide, a drug, a ligand for a    biological target, an immunoconjugate, a chemomimetic functional    group, a glycolipid, a labelling agent, an enzyme, a metal ion    chelate, an enzyme cofactor, a cytotoxic compound, a growth factor,    a hormone, a cytokine, a toxin, a prodrug, an antimetabolite, a    microtubule inhibitor, a radioactive material, a targeting moiety,    an aryl group, a linear or branched alkenyl group, a linear or    branched alkyl group, wherein said alkyl, alkenyl or aryl group is    substituted with a perfluorocarbon, perfluorocarbon-R₄,    perfluorocarbon-OR₄, or

-   R′ is hydrogen, a fluorochrome, an indole-containing compound, an    antibody or antibody fragment, a peptide, an oligonucleotide, a    drug, a ligand for a biological target, an immunoconjugate, a    chemomimetic functional group, a glycolipid, a labelling agent, an    enzyme, a metal ion chelate, an enzyme cofactor, a cytotoxic    compound, a growth factor, a hormone, a cytokine, a toxin, a    prodrug, an antimetabolite, a microtubule inhibitor, a radioactive    material, a targeting moiety, an aryl group, a linear or branched    alkenyl group, a linear or branched alkyl group, wherein said alkyl,    alkenyl or aryl group is substituted with a perfluorocarbon,    perfluorocarbon-R₄, perfluorocarbon-OR₄;-   R₄ is independently an alkyl group, an alkylene group, a carboxylate    group, a carboxylic acid group, and amino group, an ammonium group,    an alkoxyl group, a hydroxyl group or another nitrogen, oxygen or    sulfur-containing group-   A is, independently: O, S or NH-   B or B′ is, independently, alkyl, substituted alkyl, aryl,    substituted aryl, OH, NH₂, OR₁, NHR₁; Where R₁ is alkyl, substituted    alkyl, aryl, substituted aryl, wherein the said alkyl or aryl group    is either perfluorinated or substituted with perfluorinated    compound, NHR;-   n is an integer from 1-10,000-   Each m, independently, is an integer from 1-1,000;-   y or y′ independently, is an integer from 1-10.

In another embodiment, this invention provides an amphiphilic polymer,characterized by the structure of the general formula VI:

wherein

-   R is a hydroxyl (OH), O-alkyl, SH, S-alkyl, or an acid activating    group such as halogen (Cl, Br, I), O-vinyl, O-allyl, O-aryl,    OCOalkyl, OCOaryl, OCH₂CF₃NH₂, NH, SH, an acyl group, a    fluorochrome, an indole-containing compound, an antibody or antibody    fragment, a peptide, an oligonucleotide, a drug, a ligand for a    biological target, an immunoconjugate, a chemomimetic functional    group, a glycolipid, a labelling agent, an enzyme, a metal ion    chelate, an enzyme cofactor, a cytotoxic compound, a growth factor,    a hormone, a cytokine, a toxin, a prodrug, an antimetabolite, a    microtubule inhibitor, a radioactive material, a targeting moiety,    an aryl group, a linear or branched alkenyl group, a linear or    branched alkyl group, wherein said alkyl, alkenyl or aryl group is    substituted with a perfluorocarbon, perfluorocarbon-R₄,    perfluorocarbon-OR₄, or

-   R′ is hydrogen, a fluorochrome, an indole-containing compound, an    antibody or antibody fragment, a peptide, an oligonucleotide, a    drug, a ligand for a biological target, an immunoconjugate, a    chemomimetic functional group, a glycolipid, a labelling agent, an    enzyme, a metal ion chelate, an enzyme cofactor, a cytotoxic    compound, a growth factor, a hormone, a cytokine, a toxin, a    prodrug, an antimetabolite, a microtubule inhibitor, a radioactive    material, a targeting moiety, an aryl group, a linear or branched    alkenyl group, a linear or branched alkyl group, wherein said alkyl,    alkenyl or aryl group is substituted with a perfluorocarbon,    perfluorocarbon-R4, perfluorocarbon-OR4.-   R₄ is independently an alkyl group, an alkylene group, a carboxylate    group, a carboxylic acid group, and amino group, an ammonium group,    an alkoxyl group, a hydroxyl group or another nitrogen, oxygen or    sulfur-containing group;-   T, independently is:

-   z is, independently, a halogen, a nitro group, a hydroxy group, an    amino group, an alkyl group, a substituted alkyl group, an aryl    group, a substituted aryl group, wherein said substituted alkyl or    aryl group is substituted with a perfluorocarbon,    perfluorocarbon-R₄, perfluorocarbon-OR₄, or

-   A is, independently O, S, NH;-   p, p′ are integers;-   each x, independently, is an integer from 1-1000; and-   y is an integer from 1-10,000.

In another embodiment, this invention provides an amphiphilic polymer,characterized by the structure of the general formula VII:

wherein

-   R is a hydroxyl (OH), O-alkyl, SH, S-alkyl, or an acid activating    group such as halogen (Cl, Br, I), O-vinyl, O-allyl, O-aryl,    OCOalkyl, OCOaryl, OCH₂CF₃NH₂, NH, SH, an acyl group, a    fluorochrome, an indole-containing compound, an antibody or antibody    fragment, a peptide, an oligonucleotide, a drug, a ligand for a    biological target, an immunoconjugate, a chemomimetic functional    group, a glycolipid, a labelling agent, an enzyme, a metal ion    chelate, an enzyme cofactor, a cytotoxic compound, a growth factor,    a hormone, a cytokine, a toxin, a prodrug, an antimetabolite, a    microtubule inhibitor, a radioactive material, a targeting moiety,    an aryl group, a linear or branched alkenyl group, a linear or    branched alkyl group, wherein said alkyl, alkenyl or aryl group is    substituted with a perfluorocarbon, perfluorocarbon-R₄,    perfluorocarbon-OR₄, or

-   R′ is hydrogen, a fluorochrome, an indole-containing compound, an    antibody or antibody fragment, a peptide, an oligonucleotide, a    drug, a ligand for a biological target, an immunoconjugate, a    chemomimetic functional group, a glycolipid, a labelling agent, an    enzyme, a metal ion chelate, an enzyme cofactor, a cytotoxic    compound, a growth factor, a hormone, a cytokine, a toxin, a    prodrug, an antimetabolite, a microtubule inhibitor, a radioactive    material, a targeting moiety, an aryl group, a linear or branched    alkenyl group, a linear or branched alkyl group, wherein said alkyl,    alkenyl or aryl group is substituted with a perfluorocarbon,    perfluorocarbon-R₄, perfluorocarbon-OR₄,-   R₄ is independently an alkyl group, an alkylene group, a carboxylate    group, a carboxylic acid group, and amino group, an ammonium group,    an alkoxyl group, a hydroxyl group or another nitrogen, oxygen or    sulfur-containing group;-   T, independently is:

-   z is, independently, H, alkyl, aryl, NH2, NH-alkyl, NH-acyl,    NH-aryl, OH, O-acyl, O-alkyl, O-aryl, a halogen, a nitro group, a    hydroxy group, a substituted alkyl group, a substituted aryl group,    wherein said substituted alkyl or aryl group is substituted with a    perfluorocarbon, perfluorocarbon-R₄, perfluorocarbon-OR₄, or

-   A is, independently O, S, NH;-   p, p′ are integers;-   each y, independently, is an integer from 1-1000; and-   n is an integer from 1-10,000.

In one embodiment, the polymers with a structure characterized byformula I or II of this invention will be such that the weight of afraction of the polymer ranges between 0-5% or, in another embodiment,6-99% of the weight of said polymer, or, in another embodiment, 5-10% ofthe weight of said polymer, or in another embodiments, x representsabout 10-25% of the weight of said polymer, or in another embodiment, xrepresents from about 30-75% of the weight of said polymer, or inanother embodiment, x represents from about 50-100% of the weight ofsaid polymer, wherein the fraction is represented by the structure:

In one embodiment, the polymers with a structure characterized byformula I or II of this invention will be such that the weight of afraction of the polymer ranges between 1-94% or, in another embodiment,0% of the weight of said polymer, or, in another embodiment, 5-10% ofthe weight of said polymer, or in another embodiments, x representsabout 10-25% of the weight of said polymer, or in another embodiment, xrepresents from about 30-75% of the weight of said polymer, or inanother embodiment, x represents from about 50-90% of the weight of saidpolymer, wherein the fraction is represented by the structure:

The polymers of this invention are amphiphilic. In one embodiment, theterm “amphiphilic” refers to a molecule that contains both hydrophilicand lipophilic (or, synonymously, hydrophobic) moieties.

In one embodiment, the term “alkyl” refers to C₁₋₃₂ straight-chain orC₁₋₃₂ branched hydrocarbons, e.g. methyl, isobutyl, hexyl, etc. Inanother embodiment, the term “alkyl” (or “lower alkyl”) refers to both“unsubstituted alkyls” and “substituted alkyls”, the latter of whichrefers to alkyl moieties having substituents replacing a hydrogen on oneor more carbons of the hydrocarbon backbone. Such substituents caninclude, for example, a halogen, a hydroxyl, a carbonyl (such as acarboxyl, an ester, a formyl, or a ketone), a thiocarbonyl (such as athioester, a thioacetate, or a thioformate), an alkoxyl, a phosphoryl, aphosphonate, a phosphinate, an amino, an amido, an amidine, an imine, acyano, a nitro, an azido, a sulfhydryl, an alkylthio, a sulfate, asulfonate, a sulfamoyl, a sulfonamido, a sulfonyl, a heterocyclyl, anaralkyl, or an aromatic or heteroaromatic moiety. It will be understoodby those skilled in the art that the moieties substituted on thehydrocarbon chain can themselves be substituted, if appropriate. Forinstance, the substituents of a substituted alkyl may includesubstituted and unsubstituted forms of amino, azido, imino, amido,phosphoryl (including phosphonate and phosphinate), sulfonyl (includingsulfate, sulfonamido, sulfamoyl and sulfonate), and silyl groups, aswell as ethers, alkylthios, carbonyls (including ketones, aldehydes,carboxylates, and esters), —CF₃, —CN and the like.

In one embodiment, the term “alkoxy” refers to an alkyl group connectedto a main chain or backbone through an oxygen atom. In anotherembodiment, the term “alkoxyl” or “alkoxy” are interchangeable, andrepresentative alkoxyl groups include methoxy, ethoxy, propyloxy,tert-butoxy and the like.

In one embodiment, the term “aryl” refers to aromatic rings such asphenyl, pyridinyl, thienyl, thiazolyl, or furyl, optionally substitutedwith one or more groups, such as a halo group, a haloalkyl group, anamino group, or an alkyl group. In one embodiment, the term “aryl”includes 5-, 6- and 7-membered single-ring aromatic groups that mayinclude from zero to four heteroatoms, for example, benzene, pyrrole,furan, thiophene, imidazole, oxazole, thiazole, triazole, pyrazole,pyridine, pyrazine, pyridazine and pyrimidine, and the like. Those arylgroups having heteroatoms in the ring structure may also be referred toas “aryl heterocycles” or “heteroaromatics”. The aromatic ring can besubstituted at one or more ring positions with such substituents asdescribed above, for example, halogen, azide, alkyl, aralkyl, alkenyl,alkynyl, cycloalkyl, hydroxyl, amino, nitro, sulfhydryl, imino, amido,phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio,sulfonyl, sulfonamido, ketone, aldehyde, ester, heterocyclyl, aromaticor heteroaromatic moieties, —CF₃, —CN, or the like. The term “aryl” alsoincludes polycyclic ring systems having two or more rings in which twoor more carbons are common to two adjoining rings (the rings are“fused”) wherein at least one of the rings is aromatic, e.g., the otherrings can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls and/orheterocyclyls. In one embodiment, the term “aryloxy” refers to arylgroups attached to a main chain or backbone through an oxygen atom.

In one embodiment, the term “amine” refers to any amine, includingprimary, secondary, tertiary, quaternary, or a combination thereof, asapplicable herein.

In one embodiment, the term “acid activating group” refers to a groupwhich facilitates conjugation of the polymers with a desired substance,via a suitable reactive derivative of a carboxylic acid, which maycomprise inter-alia, an acyl halide, for example an acyl chloride formedby the reaction of the acid and an inorganic acid chloride, for examplethionyl chloride; a mixed anhydride, for example an anhydride formed bythe reaction of the acid and a chloroformate such as isobutylchloroformate; an active ester, for example an ester formed by thereaction of the acid and a phenol such as pentafluorophenol, an estersuch as pentafluorophenyl trifluoroacetate or an alcohol such asmethanol, ethanol, isopropanol, butanol or N-hydroxybenzotriazole; anacyl azide, for example an azide formed by the reaction of the acid andazide such as diphenylphosphoryl azide; an acyl cyanide, for example acyanide formed by the reaction of an acid and a cyanide such asdiethylphosphoryl cyanide; or the product of the reaction of the acidand a carbodiimide such as dicyclohexylcarbodiimide.

In one embodiment, the term “fluorochrome” refers to a fluorescentsubstance and may comprise, inter-alia, DAPI, FITC, Cy3, Cy3.5, Cy5,Cy5.5, Cy7, GFP, and others as will be appreciated by one skilled in theart, each selected for specific properties, for example, as described byWaggoner, A. (Methods in Enzymology 246:362-373 (1995) hereinincorporated by reference).

In one embodiment, the term “antibody or antibody fragment” refers tointact antibody molecules as well as functional fragments thereof, suchas Fab, F(ab′)2, and Fv that are capable of binding to an epitope. Inone embodiment, an Fab fragment refers to the fragment which contains amonovalent antigen-binding fragment of an antibody molecule, which canbe produced by digestion of whole antibody with the enzyme papain toyield an intact light chain and a portion of one heavy chain. In oneembodiment, Fab′ fragment refers to a part of an antibody molecule thatcan be obtained by treating whole antibody with pepsin, followed byreduction, to yield an intact light chain and a portion of the heavychain. Two Fab′ fragments may be obtained per antibody molecule. In oneembodiment, (Fab′)₂ refers to a fragment of an antibody that can beobtained by treating whole antibody with the enzyme pepsin withoutsubsequent reduction. In another embodiment, F(ab′)₂ is a dimer of twoFab′ fragments held together by two disulfide bonds. In one embodiment,Fv, may refer to a genetically engineered fragment containing thevariable region of the light chain and the variable region of the heavychain expressed as two chains. In one embodiment, the antibody fragmentmay be a single chain antibody (“SCA”), a genetically engineeredmolecule containing the variable region of the light chain and thevariable region of the heavy chain, linked by a suitable polypeptidelinker as a genetically fused single chain molecule.

Methods of making these fragments are known in the art. (See forexample, Harlow and Lane, Antibodies: A Laboratory Manual, Cold SpringHarbor Laboratory, New York, 1988, incorporated herein by reference).

In one embodiment, the antibody will recognize an epitope, which inanother embodiment, refers to antigenic determinant on an antigen towhich the paratope of an antibody binds. Epitopic determinants may, inother embodiments, consist of chemically active surface groupings ofmolecules such as amino acids or carbohydrate side chains and in otherembodiments, may have specific three dimensional structuralcharacteristics, and/or in other embodiments, have specific chargecharacteristics.

Antibody fragments according to the present invention can be prepared byproteolytic hydrolysis of the antibody or by expression in E. coli ormammalian cells (e.g. Chinese hamster ovary cell culture or otherprotein expression systems) of DNA encoding the fragment.

In other embodiments, antibody fragments can be obtained by pepsin orpapain digestion of whole antibodies by conventional methods. Forexample, antibody fragments can be produced by enzymatic cleavage ofantibodies with pepsin to provide a 5S fragment denoted F(ab′)2. Thisfragment can be further cleaved using a thiol reducing agent, andoptionally a blocking group for the sulfhydryl groups resulting fromcleavage of disulfide linkages, to produce 3.5S Fab′ monovalentfragments. Alternatively, an enzymatic cleavage using pepsin producestwo monovalent Fab′ fragments and an Fc fragment directly. These methodsare described, for example, by Goldenberg, U.S. Pat. Nos. 4,036,945 and4,331,647, and references contained therein, which patents are herebyincorporated by reference in their entirety. See also Porter, R. R.,Biochem. J., 73: 119-126, 1959. Other methods of cleaving antibodies,such as separation of heavy chains to form monovalent light-heavy chainfragments, further cleavage of fragments, or other enzymatic, chemical,or genetic techniques may also be used, so long as the fragments bind tothe antigen that is recognized by the intact antibody.

Fv fragments comprise an association of VH and VL chains. Thisassociation may be noncovalent, as described in Inbar et al., Proc.Nat'l Acad. Sci. USA 69:2659-62, 1972. Alternatively, the variablechains can be linked by an intermolecular disulfide bond or cross-linkedby chemicals such as glutaraldehyde. Preferably, the Fv fragmentscomprise VH and VL chains connected by a peptide linker. Thesesingle-chain antigen binding proteins (sFv) are prepared by constructinga structural gene comprising DNA sequences encoding the VH and VLdomains connected by an oligonucleotide. The structural gene is insertedinto an expression vector, which is subsequently introduced into a hostcell such as E. coli. The recombinant host cells synthesize a singlepolypeptide chain with a linker peptide bridging the two V domains.Methods for producing sFvs are described, for example, by Whitlow andFilpula, Methods, 2: 97-105, 1991; Bird et al., Science 242:423-426,1988; Pack et al., Bio/Technology 11:1271-77, 1993; and Ladner et al.,U.S. Pat. No. 4,946,778, which is hereby incorporated by reference inits entirety.

Another form of an antibody fragment is a peptide coding for a singlecomplementarity-determining region (CDR). CDR peptides (“minimalrecognition units”) can be obtained by constructing genes encoding theCDR of an antibody of interest. Such genes are prepared, for example, byusing the polymerase chain reaction to synthesize the variable regionfrom RNA of antibody-producing cells. See, for example, Larrick and Fry,Methods, 2: 106-10, 1991.

In one embodiment, the term “peptide” refers to native peptides (eitherdegradation products, synthetically synthesized peptides or recombinantpeptides) and/or peptidomimetics (typically, synthetically synthesizedpeptides), such as peptoids and semipeptoids which are peptide analogs,which may have, for example, modifications rendering the peptides morestable while in a body or more capable of penetrating into cells. Suchmodifications include, but are not limited to N terminus modification, Cterminus modification, peptide bond modification, including, but notlimited to, CH₂—NH, CH₂—S, CH₂—S═O, O═C—NH, CH₂—O, CH₂—CH₂, S═C—NH,CH═CH or CF═CH, backbone modifications, and residue modification.Methods for preparing peptidomimetic compounds are well known in the artand are specified, for example, in Quantitative Drug Design, C. A.Ramsden Gd., Chapter 17.2, F. Choplin Pergamon Press (1992), which isincorporated by reference as if fully set forth herein. Further detailsin this respect are provided hereinunder.

Peptide bonds (—CO—NH—) within the peptide may be substituted, forexample, by N-methylated bonds (—N(CH₃)—CO—), ester bonds(—C(R)H—C—O—O—C(R)—N—), ketomethylen bonds (—CO—CH₂—), *-aza bonds(—NH—N(R)—CO—), wherein R is any alkyl, e.g., methyl, carba bonds(—CH₂—NH—), hydroxyethylene bonds (—CH(OH)—CH₂—), thioamide bonds(—CS—NH—), olefinic double bonds (—CH═CH—), retro amide bonds (—NH—CO—),peptide derivatives (—N(R)—CH₂—CO—), wherein R is the “normal” sidechain, naturally presented on the carbon atom.

These modifications can occur at any of the bonds along the peptidechain and even at several (2-3) at the same time. Natural aromatic aminoacids, Trp, Tyr and Phe, may be substituted for synthetic non-naturalacid such as TIC, naphthylelanine (Nol), ring-methylated derivatives ofPhe, halogenated derivatives of Phe or o-methyl-Tyr.

In addition to the above, the peptides of the present invention may alsoinclude one or more modified amino acids or one or more non-amino acidmonomers (e.g. fatty acids, complex carbohydrates etc).

In one embodiment, the term “amino acid” or “amino acids” is understoodto include the 20 naturally occurring amino acids; those amino acidsoften modified post-translationally in vivo, including, for example,hydroxyproline, phosphoserine and phosphothreonine; and other unusualamino acids including, but not limited to, 2-aminoadipic acid,hydroxylysine, isodesmosine, nor-valine, nor-leucine and ornithine.Furthermore, the term “amino acid” may include both D- and L-aminoacids.

Peptides or proteins of this invention may be prepared by varioustechniques known in the art, including phage display libraries[Hoogenboom and Winter, J. Mol. Biol. 227:381 (1991); Marks et al., J.Mol. Biol. 222:581 (1991)].

In one embodiment, the term “oligonucleotide” is interchangeable withthe term “nucleic acid”, and may refer to a molecule, which may include,but is not limited to, prokaryotic sequences, eukaryotic mRNA, cDNA fromeukaryotic mRNA, genomic DNA sequences from eukaryotic (e.g., mammalian)DNA, and even synthetic DNA sequences. The term also refers to sequencesthat include any of the known base analogs of DNA and RNA.

Nucleic acid sequences, of which the polymers, micelles and/orcompositions of this invention may be comprised, may include their beinga part a particular vector, depending, in one embodiment, upon thedesired method of introduction of the sequence within cells. In oneembodiment, such vectors may be encapsulated within the micelles of thisinvention. In one embodiment, polynucleotide segments encoding sequencesof interest can be ligated into commercially available expression vectorsystems suitable for transducing/transforming mammalian cells and fordirecting the expression of recombinant products within the transducedcells. It will be appreciated that such commercially available vectorsystems can easily be modified via commonly used recombinant techniquesin order to replace, duplicate or mutate existing promoter or enhancersequences and/or introduce any additional polynucleotide sequences suchas for example, sequences encoding additional selection markers orsequences encoding reporter polypeptides.

The efficacy of a particular expression vector system and method ofintroducing nucleic acid into a cell can be assessed by standardapproaches routinely used in the art. For example, DNA introduced into acell can be detected by a filter hybridization technique (e.g., Southernblotting) and RNA produced by transcription of introduced DNA can bedetected, for example, by Northern blotting, RNase protection or reversetranscriptase-polymerase chain reaction (RT-PCR). The gene product canbe detected by an appropriate assay, for example by immunologicaldetection of a produced protein, such as with a specific antibody, or bya functional assay to detect a functional, activity of the gene product,such as an enzymatic assay. If the gene product of interest to beexpressed by a cell is not readily assayable, an expression system canfirst be optimized using a reporter gene linked to the regulatoryelements and vector to be used. The reporter gene encodes a geneproduct, which is easily detectable and, thus, can be used to evaluateefficacy of the system. Standard reporter genes used in the art includegenes encoding β-galactosidase, chloramphenicol acetyl transferase,luciferase and human growth hormone, or any of the marker proteinslisted herein.

As will be appreciated by one skilled in the art, a fragment orderivative of a nucleic acid sequence or gene that encodes for a proteinor peptide can still function in the same manner as the entire, wildtype gene or sequence. Likewise, forms of nucleic acid sequences canhave variations as compared to wild type sequences, neverthelessencoding the protein or peptide of interest, or fragments thereof,retaining wild type function exhibiting the same biological effect,despite these variations. Each of these represents a separate embodimentof this present invention.

The nucleic acids can be produced by any synthetic or recombinantprocess such as is well known in the art. Nucleic acids can further bemodified to alter biophysical or biological properties by means oftechniques known in the art. For example, the nucleic acid can bemodified to increase its stability against nucleases (e.g.,“end-capping”), or to modify its lipophilicity, solubility, or bindingaffinity to complementary sequences.

DNA according to the invention can also be chemically synthesized bymethods known in the art. For example, the DNA can be synthesizedchemically from the four nucleotides in whole or in part by methodsknown in the art. Such methods include those described in Caruthers(1985). DNA can also be synthesized by preparing overlappingdouble-stranded oligonucleotides, filling in the gaps, and ligating theends together (see, generally, Sambrook et al. (1989) and Glover et al.(1995)). DNA expressing functional homologues of the protein can beprepared from wild-type DNA by site-directed mutagenesis (see, forexample, Zoller et al. (1982); Zoller (1983); and Zoller (1984);McPherson (1991)). The DNA obtained can be amplified by methods known inthe art. One suitable method is the polymerase chain reaction (PCR)method described in Saiki et al. (1988), Mullis et al., U.S. Pat. No.4,683,195, and Sambrook et al. (1989).

Methods for modifying nucleic acids to achieve specific purposes aredisclosed in the art, for example, in Sambrook et al. (1989). Moreover,the nucleic acid sequences of the invention can include one or moreportions of nucleotide sequence that are non-coding for the protein ofinterest. Variations in the DNA sequences, which are caused by pointmutations or by induced modifications (including insertion, deletion,and substitution) to enhance the activity, half-life or production ofthe polypeptides encoded thereby, are also encompassed in the invention.

In another embodiment, the agent that inhibits gene expression, activityor function comprises a nucleic acid. The nucleic acid may, in oneembodiment, be DNA, or in another embodiment, the nucleic acid is RNA.In other embodiments, the nucleic acid may be single or double stranded.

In another embodiment, the agent is a nucleic acid that is antisense inorientation to a sequence encoding for a caspase.

In one embodiment, the polymers, micelles or compositions of thisinvention may be used for gene silencing applications. In oneembodiment, the activity or function of a particular gene is suppressedor diminished, via the use of antisense oligonucleotides, which arechimeric molecules, containing two or more chemically distinct regions,each made up of at least one nucleotide. In one embodiment, theantisense molecules may be conjugated to the polymers of this invention,as described, or in another embodiment, encapsulated within micelles ofthis invention, much as any of the respective groups listed herein,applicable in the methods of this invention, in another embodiment, maybe conjugated to the polymers of this invention, or encapsulated withinmicelles of this invention.

Antisense oligonucleotides, in one embodiment, may be chimericoligonucleotides, which contain at least one region wherein theoligonucleotide is modified so as to confer upon the oligonucleotide anincreased resistance to nuclease degradation, increased cellular uptake,and/or increased binding affinity for the target polynucleotide. Anadditional region of the oligonucleotide may serve as a substrate forenzymes capable of cleaving RNA:DNA or RNA:RNA hybrids, which accordingto this aspect of the invention, serves as a means of gene silencing viadegradation of specific sequences. Cleavage of the RNA target can beroutinely detected by gel electrophoresis and, if necessary, associatednucleic acid hybridization techniques known in the art.

The chimeric antisense oligonucleotides may, in one embodiment, beformed as composite structures of two or more oligonucleotides and/ormodified oligonucleotides, as is well described in the art (see, forexample, U.S. Pat. Nos. 5,013,830; 5,149,797; 5,220,007; 5,256,775;5,366,878; 5,403,711; 5,491,133; 5,565,350; 5,623,065; 5,652,355;5,652,356; and 5,700,922), and can, in another embodiment, comprise aribozyme sequence.

Inhibition of gene expression, activity or function is effected, inanother embodiment, via the use of small interfering RNAs, whichprovides sequence-specific inhibition of gene expression. Administrationof double stranded/duplex RNA (dsRNA) corresponding to a single gene inan organism can silence expression of the specific gene by rapiddegradation of the mRNA in affected cells. This process is referred toas gene silencing, with the dsRNA functioning as a specific RNAinhibitor (RNAi). RNAi may be derived from natural sources, such as inendogenous virus and transposon activity, or it can be artificiallyintroduced into cells (Elbashir S M, et al (2001). Nature 411:494-498)via microinjection (Fire et al. (1998) Nature 391: 806-11), or bytransformation with gene constructs generating complementary RNAs orfold-back RNA, or by other vectors (Waterhouse, P. M., et al. (1998).Proc. Natl. Acad. Sci. USA 95, 13959-13964 and Wang, Z., et al. (2000).J. Biol. Chem. 275, 40174-40179). The RNAi mediating mRNA degradation,in one embodiment, comprises duplex or double-stranded RNA, or, in otherembodiments, include single-stranded RNA, isolated RNA (partiallypurified RNA, essentially pure RNA, synthetic RNA, recombinantlyproduced RNA), as well as altered RNA that differs from naturallyoccurring RNA by the addition, deletion and/or alteration of one or morenucleotides.

When referring to nucleic acid sequences utilized as modulators in thisinvention, it is to be understood that such reference allows for theincorporation of non-nucleotide material, which may be added, forexample, to the end(s) of the nucleotide sequence, including forexample, terminal 3′ hydroxyl groups, or internal additions, at one ormore nucleotides. Nucleic acids may, in another embodiment, incorporatenon-standard nucleotides, including non-naturally-occurring nucleotides.Alterations may also include the construction of blunt and/oroverhanging ends. Collectively all such altered nucleic acids may bereferred to as analogs, and represent contemplated embodiments of theinvention.

In another embodiment, gene expression can be inhibited/downregulatedsimply by “knocking out” the gene. Typically this is accomplished bydisrupting the gene, the promoter regulating the gene or sequencesbetween the promoter and the gene. Such disruption can be specificallydirected to a particular gene by homologous recombination where a“knockout construct” contains flanking sequences complementary to thedomain to which the construct is targeted. Insertion of the knockoutconstruct (e.g. into the gene of interest) results in disruption of thatgene. The phrases “disruption of the gene” and “gene disruption” referto insertion of a nucleic acid sequence into one region of the nativeDNA sequence (in some embodiments, in one or more exons) and/or thepromoter region of a gene so as to decrease or prevent expression ofthat gene in the cell as compared to the wild-type or naturallyoccurring sequence of the gene.

Knockout constructs can be produced by standard methods known to thoseof skill in the art. The knockout construct can be chemicallysynthesized or assembled, e.g., using recombinant DNA methods. The DNAsequence to be used in producing the knockout construct is digested witha particular restriction enzyme selected to cut at a location(s) suchthat a new DNA sequence encoding a marker gene can be inserted in theproper position within this DNA sequence. The proper position for markergene insertion is that which will serve to prevent expression of thenative gene; this position will depend on various factors such as therestriction sites in the sequence to be cut, and whether an exonsequence or a promoter sequence, or both is (are) to be interrupted(i.e., the precise location of insertion necessary to inhibit promoterfunction or to inhibit synthesis of the native exon).

It is to be understood that the above nucleic acids may be delivered toany tissue or cells in one embodiment, in their native form, or, inanother embodiment within an expression vector that is competent totransfect cells in vitro and/or in vivo, and comprise an embodiment ofthis invention.

In another embodiment, this invention provides a method of nucleic aciddelivery, comprising contacting a cell with a polymer, micelle orcomposition of this invention, comprising a nucleic acid of interest. Inone embodiment, the nucleic acid encodes for a compound, whichstimulates organogenesis, for example, the compound is osteogenic,chondrogenic or angiogenic. In another embodiment, the nucleic acidencodes for an antibacterial, antiviral, antifungal or antiparasiticpeptide or protein. In another embodiment, the nucleic acid encodes fora peptide or protein with cytotoxic or anti-cancer activity. In anotherembodiment, the nucleic acid encodes for an enzyme, a receptor, achannel protein, a hormone, a cytokine or a growth factor. In anotherembodiment, the nucleic acid encodes for a peptide or protein, which isimmunostimulatory. In another embodiment, the nucleic acid encodes for apeptide or protein, which inhibits inflammatory or immune responses. Inanother embodiment, release of the nucleic acid occurs over a period oftime.

In one embodiment, the polymers, micelles or compositions of thisinvention are targeted to cells. In one embodiment, the cell may be anyresponsive cell, such as, in one embodiment, an epithelial cell, a lungcell, a kidney cell, a liver cell, a cardiocyte, an astrocyte, a glialcell, a prostate cell, a professional antigen presenting cell, alymphocyte, an M cell, a pancreatic cell, a stem cell, a myoblast, ahepatocyte, an osteoblast, an osteocyte, an osteoclast, a chondrocyte, achodroblast, or other bone or cartilage cells and may be used forapplications as described in, for example, Wilson, J. M et al. (1988)Proc. Natl. Acad. Sci. USA 85:3014-3018; Armentano, D. et al. (1990)Proc. Natl. Acad. Sci. USA 87:6141-6145; Wolff, J. A. et al. (1990)Science 247:1465-1468; Chowdhury, J. R. et al. (1991) Science254:1802-1805; Ferry, N. et al. (1991) Proc. Natl. Acad. Sci. USA88:8377-8381; Wilson, J. M. et al. (1992) J. Biol. Chem. 267:963-967;Quantin, B. et al. (1992) Proc. Natl. Acad. Sci. USA 89:2581-2584; Dai,Y. et al. (1992) Proc. Natl. Acad. Sci. USA 89:10892-10895; vanBeusechem, V. W. et al. (1992) Proc. Natl. Acad Sci. USA 89:7640-7644;Rosenfeld, M. A. et al. (1992) Cell 68:143-155; Kay, M. A. et al. (1992)Human Gene Therapy 3:641-647; Cristiano, R. J. et al. (1993) Proc. Natl.Acad Sci. USA 90:2122-2126; Hwu, P. et al. (1993) J. Immunol.150:4104-4115; and Herz, J. and Gerard, R. D. (1993) Proc. Natl. AcadSci. USA 90:2812-2816.

In one embodiment, the polymers, micelles or compositions of thisinvention comprise a drug. In one embodiment, the term “drug” refers toa substance applicable for use in the diagnosis, or in anotherembodiment, cure, or in another embodiment, mitigation, or in anotherembodiment, treatment, or in another embodiment, prevention of adisease, disorder, condition or infection. In one embodiment, the term“drug” refers to any substance which affect the structure or function ofthe target to which it is applied.

In another embodiment, the term “drug” refers to a molecule thatalleviates a symptom of a disease or disorder when administered to asubject afflicted thereof. In one embodiment, a drug is a syntheticmolecule, or in another embodiment, a drug is a naturally occurringcompound isolated from a source found in nature.

In one embodiment, drugs may comprise antihypertensives,antidepressants, antianxiety agents, anticlotting agents,anticonvulsants, blood glucose-lowering agents, decongestants,antihistamines, antitussives, anti-inflammatories, antipsychotic agents,cognitive enhancers, cholesterol-reducing agents, antiobesity agents,autoimmune disorder agents, anti-impotence agents, antibacterial andantifungal agents, hypnotic agents, anti-Parkinsonism in agents,antibiotics, antiviral agents, anti-neoplastics, barbituates, sedatives,nutritional agents, beta blockers, emetics, anti-emetics, diuretics,anticoagulants, cardiotonics, androgens, corticoids, anabolic agents,growth hormone secretagogues, anti-infective agents, coronaryvasodilators, carbonic anhydrase inhibitors, antiprotozoals,gastrointestinal agents, serotonin antagonists, anesthetics,hypoglycemic agents, dopaminergic agents, anti-Alzheimer's Diseaseagents, anti-ulcer agents, platelet inhibitors and glycogenphosphorylase inhibitors.

In one embodiment, examples of the drugs conjugated to the polymers ofthis invention, or in another embodiment, encapsulated within a micelleof this invention, comprise, inter-alia, antihypertensives includingprazosin, nifedipine, trimazosin, amlodipine, and doxazosin mesylate;the antianxiety agent hydroxyzine; a blood glucose lowering agent suchas glipizide; an anti-impotence agent such as sildenafil citrate;anti-neoplastics such as chlorambucil, lomustine or echinomycin;anti-inflammatory agents such as betamethasone, prednisolone, piroxicam,aspirin, flurbiprofen and(+)-N-{4-[3-(4-fluorophenoxy)phenoxy]-2-cyclopenten-1-yl}-N-hyroxyurea;antivirals such as acyclovir, nelfinavir, or virazole;vitamins/nutritional agents such as retinol and vitamin E; emetics suchas apomorphine; diuretics such as chlorthalidone and spironolactone; ananticoagulant such as dicumarol; cardiotonics such as digoxin anddigitoxin; androgens such as 17-methyltestosterone and testosterone; amineral corticoid such as desoxycorticosterone; a steroidalhypnotic/anesthetic such as alfaxalone; an anabolic agent such asfluoxymesterone or methanstenolone; antidepression agents such asfluoxetine, pyroxidine, venlafaxine, sertraline, paroxetine, sulpiride,[3,6-dimethyl-2-(2,4,6-trimethyl-phenoxy)-pyridin-4-yl]-(lethylpropyl)-amineor 3,5-dimethyl-4-(3′-pentoxy)-2-(2′,4′,6′-trimethylphenoxy)pyridine; anantibiotic such as ampicillin and penicillin G; an anti-infective suchas benzalkonium chloride or chlorhexidine; a coronary vasodilator suchas nitroglycerin or mioflazine; a hypnotic such as etomidate; a carbonicanhydrase inhibitor such as acetazolamide or chlorzolamide; anantifungal such as econazole, terconazole, fluconazole, voriconazole orgriseofulvin; an antiprotozoal such as metronidazole; an imidazole-typeanti-neoplastic such as tubulazole; an anthelmintic agent such asthiabendazole or oxfendazole; an antihistamine such as astemizole,levocabastine, cetirizine, or cinnarizine; a decongestant such aspseudoephedrine; antipsychotics such as fluspirilene, penfluridole,risperidone or ziprasidone; a gastrointestinal agent such as loperamideor cisapride; a serotonin antagonist such as ketanserin or mianserin; ananesthetic such as lidocaine; a hypoglycemic agent such asacetohexamide; an anti-emetic such as dimenhydrinate; an antibacterialsuch as cotrimoxazole; a dopaminergic agent such as L-DOPA;anti-Alzheimer agents such as THA or donepezil; an anti-ulcer agent/H2antagonist such as famotidine; a sedative/hypnotic such aschlordiazepoxide or triazolam; a vasodilator such as alprostadil; aplatelet inhibitor such as prostacyclin; an ACEinhibitor/antihypertensive such as enalaprilic acid or lisinopril; atetracycline antibiotic such as oxytetracycline or minocycline; amacrolide antibiotic such as azithromycin, clarithromycin, erythromycinor spiramycin; and glycogen phosphorylase inhibitors such as[R—(R*S*)]-5-chloro-N-[2-hydroxy-3{methoxymethylamino}-3-oxo-1-(phenylmethyl)-propyl]-1H-indole-2-carboxamideor 5-chloro-1-Hindole-2-carboxylic acid[(IS)-benzyl(2R)-hydroxy-3-((3R,4S)dihydroxy-pyrrolidin-1-yl-)-oxypropyl]amide.

Further examples of drugs deliverable by the invention are theglucose-lowering drug chlorpropamide, the anti-fungal fluconazole, theanti-hypercholesterolemic atorvastatin calcium, the antipsychoticthiothixene hydrochloride, the anxiolytics hydroxyzine hydrochloride ordoxepin hydrochloride, the anti-hypertensive amlodipine besylate, theantiinflammatories piroxicam and celicoxib and valdicoxib, and theantibiotics carbenicillin indanyl sodium, bacampicillin hydrochloride,troleandomycin, and doxycycline hyclate.

In another embodiment a drug of this invention may comprise otherantineoplastic agents such as platinum compounds (e.g., spiroplatin,cisplatin, and carboplatin), methotrexate, fluorouracil, adriamycin,mitomycin, ansamitocin, bleomycin, cytosine arabinoside, arabinosyladenine, mercaptopolylysine, vincristine, busulfan, chlorambucil,melphalan (e.g., PAM, L-PAM or phenylalanine mustard), mercaptopurine,mitotane, procarbazine hydrochloride dactinomycin (actinomycin D),daunorubicin hydrochloride, doxorubicin hydrochloride, paclitaxel andother taxenes, rapamycin, manumycin A, TNP-470, plicamycin(mithramycin), aminoglutethimide, estramustine phosphate sodium,flutamide, leuprolide acetate, megestrol acetate, tamoxifen citrate,testolactone, trilostane, amsacrine (m-AMSA), asparaginase(L-asparaginase) Erwina asparaginase, interferon .alpha.-2a, interferon.alpha.-2b, teniposide (VM-26), vinblastine sulfate (VLB), vincristinesulfate, bleomycin sulfate, hydroxyurea, procarbazine, and dacarbazine;mitotic inhibitors such as etoposide, colchicine, and the vincaalkaloids, radiopharmaceuticals such as radioactive iodine andphosphorus products; hormones such as progestins, estrogens andantiestrogens; anti-helmintics, antimalarials, and antituberculosisdrugs; biologicals such as immune serums, antitoxins and antivenoms;rabies prophylaxis products; bacterial vaccines; viral vaccines;respiratory products such as xanthine derivatives theophylline andaminophylline; thyroid agents such as iodine products and anti-thyroidagents; cardiovascular products including chelating agents and mercurialdiuretics and cardiac glycosides; glucagon; blood products such asparenteral iron, hemin, hematoporphyrins and their derivatives;biological response modifiers such as muramyldipeptide,muramyltripeptide, microbial cell wall components, lymphokines (e.g.,bacterial endotoxin such as lipopolysaccharide, macrophage activationfactor), sub-units of bacteria (such as Mycobacteria, Corynebacteria),the synthetic dipeptide N-acetyl-muramyl-L-alanyl-D-isoglutamine;anti-fungal agents such as ketoconazole, nystatin, griseofulvin,flucytosine (5-fc), miconazole, amphotericin B, ricin, cyclosporins, andβ-lactam antibiotics (e.g., sulfazecin); hormones such as growthhormone, melanocyte stimulating hormone, estradiol, beclomethasonedipropionate, betamethasone, betamethasone acetate and betamethasonesodium phosphate, vetamethasone disodium phosphate, vetamethasone sodiumphosphate, cortisone acetate, dexamethasone, dexamethasone acetate,dexamethasone sodium phosphate, flunisolide, hydrocortisone,hydrocortisone acetate, hydrocortisone cypionate, hydrocortisone sodiumphosphate, hydrocortisone sodium succinate, methylprednisolone,methylprednisolone acetate, methylprednisolone sodium succinate,paramethasone acetate, prednisolone, prednisolone acetate, prednisolonesodium phosphate, prednisolone tebutate, prednisone, triamcinolone,triamcinolone acetonide, triamcinolone diacetate, triamcinolonehexacetonide, fludrocortisone acetate, oxytocin, vassopressin, and theirderivatives; vitamins such as cyanocobalamin neinoic acid, retinoids andderivatives such as retinol palmitate, and .alpha.-tocopherol; peptides,such as manganese super oxide dismutase; enzymes such as alkalinephosphatase; anti-allergic agents such as amelexanox; anti-coagulationagents such as phenprocoumon and heparin; circulatory drugs such aspropranolol; metabolic potentiators such as glutathione; antitubercularssuch as para-aminosalicylic acid, isoniazid, capreomycin sulfatecycloserine, ethambutol hydrochloride ethionamide, pyrazinamide,rifampin, and streptomycin sulfate; antivirals such as amantadineazidothymidine (AZT, DDI, Foscarnet, or Zidovudine), ribavirin andvidarabine monohydrate (adenine arabinoside, ara-A); antianginals suchas diltiazem, nifedipine, verapamil, erythritol tetranitrate, isosorbidedinitrate, nitroglycerin (glyceryl trinitrate) and pentaerythritoltetranitrate; anticoagulants such as phenprocoumon, heparin; antibioticssuch as dapsone, chloramphenicol, neomycin, cefaclor, cefadroxil,cephalexin, cephradine erythromycin, clindamycin, lincomycin,amoxicillin, ampicillin, bacampicillin, carbenicillin, dicloxacillin,cyclacillin, picloxacillin, hetacillin, methicillin, nafcillin,oxacillin, penicillin including penicillin G and penicillin V,ticarcillin rifampin and tetracycline; antiinflammatories such asdiflunisal, ibuprofen, indomethacin, meclofenamate, mefenamic acid,naproxen, oxyphenbutazone, phenylbutazone, piroxicam, sulindac,tolmetin, aspirin and salicylates; antiprotozoans such as chloroquine,hydroxychloroquine, metronidazole, quinine and meglumine antimonate;antirheumatics such as penicillamine; narcotics such as paregoric;opiates such as codeine, heroin, methadone, morphine and opium; cardiacglycosides such as deslanoside, digitoxin, digoxin, digitalin anddigitalis; neuromuscular blockers such as atracurium mesylate, gallaminetriethiodide, hexafluorenium bromide, metocurine iodide, pancuroniumbromide, succinylcholine chloride (suxamethonium chloride), tubocurarinechloride and vecuronium bromide; sedatives (hypnotics) such asamobarbital, amobarbital sodium, aprobarbital, butabarbital sodium,chloral hydrate, ethchlorvynol, ethinamate, flurazepam hydrochloride,glutethimide, methotrimeprazine hydrochloride, methyprylon, midazolamhydrochloride, paraldehyde, pentobarbital, pentobarbital sodium,phenobarbital sodium, secobarbital sodium, talbutal, temazepam andtriazolam; local anesthetics such as bupivacaine hydrochloride,chloroprocaine hydrochloride, etidocaine hydrochloride, lidocainehydrochloride, mepivacaine hydrochloride, procaine hydrochloride andtetracaine hydrochloride; general anesthetics such as droperidol,etomidate, fentanyl citrate with droperidol, ketamine hydrochloride,methohexital sodium and thiopental sodium; and radioactive particles orions such as strontium, iodide rhenium and yttrium.

In one embodiment, the term “drug” refers to a therapeutic compound. Inone embodiment, the therapeutic compound is a peptide, a protein or anucleic acid. In another embodiment, the therapeutic compound isorganogenic, such as osteogenic, chondrogenic or angiogenic. In anotherembodiment, the therapeutic compound is an antibacterial, antiviral,antifungal or antiparasitic compound. In another embodiment, thetherapeutic compound has cytotoxic or anti-cancer activity. In anotherembodiment, the therapeutic compound is an enzyme, a receptor, a channelprotein, a hormone, a cytokine or a growth factor. In anotherembodiment, the therapeutic compound is immunostimulatory. In anotherembodiment, the therapeutic compound inhibits inflammatory or immuneresponses.

In one embodiment, the term “therapeutic”, refers to a molecule, whichwhen provided to a subject in need, provides a beneficial effect. Insome cases, the molecule is therapeutic in that it functions to replacean absence or diminished presence of such a molecule in a subject. Inone embodiment, the molecule is a nucleic acid coding for the expressionof a protein is absent, such as in cases of an endogenous null mutantbeing compensated for by expression of the foreign protein. In otherembodiments, the endogenous protein is mutated, and produces anon-functional protein, compensated for by the expression of aheterologous functional protein. In other embodiments, expression of aheterologous protein is additive to low endogenous levels, resulting incumulative enhanced expression of a given protein. In other embodiments,the molecule stimulates a signalling cascade that provides forexpression, or secretion, or others of a critical element for cellularor host functioning. In one embodiment, the therapeutic compound is aprotein or polypeptide.

In one embodiment, the therapeutic protein may include cytokines, suchas interferons or interleukins, or their receptors. Lack of expressionof cytokines, or of the appropriate ones, has been implicated insusceptibility to diseases, and enhanced expression may lead toresistance to a number of infections. Expression patterns of cytokinesmay be altered to produce a beneficial effect, such as for example, abiasing of the immune response toward a Th1 type expression pattern, ora Th2 pattern in infection, or in autoimmune disease, wherein alteredexpression patterns may prove beneficial to the host.

In another embodiment, the therapeutic protein may comprise an enzyme,such as one involved in glycogen storage or breakdown. In anotherembodiment, the therapeutic protein comprises a transporter, such as anion transporter, for example CFTR, or a glucose transporter, or othertransporters whose deficiency, or inappropriate expression, results in avariety of diseases.

In another embodiment, the therapeutic protein comprises a tumorsuppressor, or pro-apoptotic compound, which alters progression ofcancer-related events.

In another embodiment, the therapeutic compound of the present inventionmay comprise an immunomodulating protein. In one embodiment, theimmunomodulating protein comprises cytokines, chemokines, complement orcomponents, such as interleukins 1 to 15, interferons alpha, beta orgamma, tumour necrosis factor, granulocyte-macrophage colony stimulatingfactor (GM-CSF), macrophage colony stimulating factor (M-CSF),granulocyte colony stimulating factor (G-CSF), chemokines such asneutrophil activating protein (NAP), macrophage chemoattractant andactivating factor (MCAF), RANTES, macrophage inflammatory peptidesMIP-1a and MIP-1b, or complement components.

In another embodiment, a therapeutic compound of this invention maycomprise a growth factor, or tissue-promoting factor. In one embodiment,the therapeutic compound is a bone morphogenetic protein, or OP-1, OP-2,BMP-5, BMP-6, BMP-2, BMP-3, BMP-4, BMP-9, DPP, Vg-1, 60A, or Vgr-1. Inanother embodiment, the therapeutic compound facilitates nerveregeneration or repair, and may include NGF, or other growth factors.

In one embodiment, drug may also refer to a nucleic acid, or constructcomprising a nucleic acid, whose expression ameliorates or abrogatessymptoms of a disease or a disorder, or diminishes, suppresses orinhibits a disease, disorder or condition. In one embodiment, thenucleic acid or construct comprising the same, is used for gene therapy,for providing or replacing endogenous expression, or in anotherembodiment, suppressing endogenous expression.

In another embodiment, the therapeutic molecule may be natural ornon-natural insulins, amylases, proteases, lipases, kinases,phosphatases, glycosyl transferases, trypsinogen, chymotrypsinogen,carboxypeptidases, hormones, ribonucleases, deoxyribonucleases,triacylglycerol lipase, phospholipase A2, elastases, amylases, bloodclotting factors, UDP glucuronyl transferases, ornithinetranscarbamoylases, cytochrome p450 enzymes, adenosine deaminases, serumthymic factors, thymic humoral factors, thymopoietins, growth hormones,somatomedins, costimulatory factors, antibodies, colony stimulatingfactors, erythropoietin, epidermal growth factors, hepaticerythropoietic factors (hepatopoietin), liver-cell growth factors,interleukins, interferons, negative growth factors, fibroblast growthfactors, transforming growth factors of the α family, transforminggrowth factors of the β family, gastrins, secretins, cholecystokinins,somatostatins, serotonins, substance P, transcription factors orcombinations thereof.

In one embodiment, the polymers, micelles or compositions of thisinvention may further comprise a ligand for a biological target, whichin another embodiment, provides for directional specificity as to whichcells or tissues are provided the polymers, micelles or compositions ofthis invention. In one embodiment, the term “ligand for a biologicaltarget” refers to a molecule which enables the specific delivery of thepolymer, micelle or composition of this invention to a particular sitein vivo. In one embodiment, such a ligand may be referred to as an“anti-receptor”, which functions to direct the polymer or micelle to,for example, virally infected cells, via anti-receptor binding to viralproteins expressed on infected cell surfaces. In this case,antireceptors to promote fusion with virally-infected cells, willrecognize and bind to virally expressed surface proteins. For example,HIV-1 infected cells may express HIV-associated proteins, such as gp120,and therefore the presence of CD4 on the polymer or micelle surfacepromotes targeting to HIV infected cells, via CD4-gp120 interaction.

The anti-receptor proteins or polypeptide fragments thereof may bedesigned to enhance fusion with cells infected with members of thefollowing viral families: Arenaviridae, Bunyaviridae, Coronaviridae,Filoviridae, Flaviviridae, Herpesviridae, Hepadnaviridae,Orthomyxoviridae, Paramyxoviridae, Poxviridae, Retroviridae, andRhabdoviridae. Additional viral targeting agents may be derived from thefollowing: African Swine Fever Virus, Boma Disease Virus, Hepatitis X,HIV-1, Human T Lymphocyte virus type-I (HTLV-1), HTLV-2, 1 5lentiviruses, Epstein-Barr Virus, papilloma viruses, herpes simplexviruses, hepatitis B and hepatitis C.

In another embodiment, targeting virally-infected cells may beaccomplished through the additional expression of viral co-receptors onan exposed surface of the polymers/micelles of this invention, forenhanced fusion facilitation with infected cells. In one embodiment, thepolymers/micelles of this invention comprise an HIV co-receptor such asCXCR4 or CCR5, for example.

Bacterial proteins expressed during intracellular infection are alsopotential targets contemplated for therapeutic intervention bypolymers/micelles of this invention. The intracellular bacteria mayinclude, amongst others: Shigella, Salmonella, Legionella, Streptococci,Mycobacteria, Francisella and Chlamydiae (See G. L. Mandell,“Introduction to Bacterial Disease” IN CECIL TEXTBOOK OF MEDICINE, (W.B.Saunders Co., 1996) 1556-7). These bacteria would be expected to expressa bacteria-related protein on the surface of the infected cell to whichthe polymers/micelles of this invention would attach.

In another embodiment, the targeting moieties may include integrins orclass II molecules of the MHC, which may be upregulated on infectedcells such as professional antigen presenting cells.

Proteins of parasitic agents, which reside intracellularly, also aretargets contemplated for targeting by the polymers/micelles of thisinvention. The intracellular parasites contemplated include for example,Protozoa. Protozoa, which infect cells, include: parasites of the genusPlasmodium (e.g., Plasmodium falciparum, P. Vivax, P. ovale and P.malariae), Trypanosoma, Toxoplasma, Leishmania, and Cryptosporidium.

Diseased and/or abnormal cells may be targeted using thepolymers/micelles of this invention by the methods described above. Thediseased or abnormal cells contemplated include: infected cells,neoplastic cells, pre-neoplastic cells, inflammatory foci, benign tumorsor polyps, cafe au lait spots, leukoplakia, other skin moles,self-reactive cells, including T and/or NK cells, etc. Any cell, towhich specific delivery of an agent to modulate its activity iscontemplated for the methods of this invention, and represents anembodiment thereof.

The polymers/micelles of this invention may be targeted using ananti-receptor that will recognize and bind to its cognate receptor orligand expressed on a diseased or abnormal cell, in another embodiment.

In one embodiment, the targeting agent specifically binds, orpreferentially binds only diseased cells, for delivery of a therapeuticagent, or in another embodiment, a cytotoxic agent. In one embodiment,the targeting agent is an antibody, or fragment thereof. Examples ofantibodies include those antibodies, which react with malignantprostatic epithelium but not with benign prostate tissue (e.g., ATCC No.HB-9119; ATCC HB-9120; and ATCC No. HB-1 1430) or react with malignantbreast cancer cells but not with normal breast tissue (e.g., ATCC No.HB-8691; ATCC No. HB-10807; and 21HB-108011). Other antibodies orfragments thereof, which react with diseased tissue and not with normaltissue, would be apparent to the skilled artisan.

A wide variety of tumor-specific antibodies are known in the art, suchas those described in U.S. Pat. Nos. 6,197,524, 6,191,255, 6,183,971,6,162,606, 6,160,099, 6,143,873, 6,140,470, 6,139,869, 6,113,897,6,106,833, 6,042,829, 6,042,828, 6,024,955, 6,020,153, 6,015,680,5,990,297, 5,990,287, 5,972,628, 5,972,628, 5,959,084, 5,951,985,5,939,532, 5,939,532, 5,939,277, 5,885,830, 5,874,255, 5,843,708,5,837,845, 5,830,470, 5,792,616, 5,767,246, 5,747,048, 5,705,341,5,690,935, 5,688,657, 5,688,505, 5,665,854, 5,656,444, 5,650,300,5,643,740, 5,635,600, 5,589,573, 5,576,182, 5,552,526, 5,532,159,5,525,337, 5,521,528, 5,519,120, 5,495,002, 5,474,755, 5,459,043,5,427,917, 5,348,880, 5,344,919, 5,338,832, 5,298,393, 5,331,093,5,244,801, and 5,169,774. See also The Monoclonal Antibody Index Volume1: Cancer (3rd edition). Accordingly, the polymers, micelles and/orcompositions of this invention may comprise tumor-specific antibodieswhich may recognize tumors derived from a wide variety of tissue types,including, but not limited to, breast, prostate, colon, lung, pharynx,thyroid, lymphoid, lymphatic, larynx, esophagus, oral mucosa, bladder,stomach, intestine, liver, pancreas, ovary, uterus, cervix, testes,dermis, bone, blood and brain.

In another embodiment, the polymers, micelles or compositions of thisinvention will incorporate an antibody which possesses tumoricidalactivity. Antibodies that possess tumoricidal activity are also known inthe art, including IMC-C225, EMD 72000, OvaRex Mab B43.13,anti-ganglioside G(D2) antibody ch 14.18, CO17-1A, trastuzumab, rhuMAbVEGF, sc-321, AF349, BAF349, AF743, BAF743, MAB743, AB1875,Anti-Flt-4AB3127, FLT41-A, rituximab, 2C3, CAMPATH 1H, 2G7, Alpha IR-3,ABX-EGF, MDX-447, anti-p75 IL-2R, anti-p64 IL-2R, and 2A11.

Epitopes to which tumor-specific antibodies bind are also well known inthe art. For example, epitopes bound by the tumor-specific antibodies ofthe invention include, but are not limited to, those known in the art tobe present on CA-125, gangliosides G(D2), G(M2) and G(D3), CD20, CD52,CD33, Ep-CAM, CEA, bombesin-like peptides, PSA, HER2/neu, epidermalgrowth factor receptor, erbB2, erbB3, erbB4, CD44v6, Ki-67,cancer-associated mucin, VEGF, VEGFRs (e.g., VEGFR3), estrogenreceptors, Lewis-Y antigen, TGF/31, IGF-1 receptor, EGFa, c-Kitreceptor, transferrin receptor, IL-2R and CO17-1A. It is to beunderstood that antibodies to these, and other epitopes, may be designedby methods well known in the art, such as, for example, as described inHarlow and Lane (1988) Antibodies: A Laboratory Manual, Cold SpringHarbor Laboratory, New York (Harlow and Lane, 1988), or “CurrentProtocols in Immunology” (Coligan, 1991), and may be incorporated in thepolymers, micelles and/or compositions of this invention, and representsembodiments thereof.

In one embodiment, the targeting moiety is a peptide, an antibody, anantibody fragment, a receptor, Protein A, Protein G, biotin, avidin,streptavidin, a metal ion chelate, an enzyme cofactor, a nucleic acid ora ligand.

In another embodiment, the targeting moiety is a peptide which binds toan underglycosylated mucin-1 protein. In one embodiment, the peptide isan EPPT1 peptide.

Mucin-1 (MUC-1) is a transmembrane molecule, which is overexpressed onthe cell surface and in intracellular compartments of almost all humanepithelial cell adenocarcinomas, including more than 90% of human breastcancers, ovarian, pancreatic, colorectal, lung, prostate, colon andgastric carcinomas. Expression has been demonstrated in non-epithelialcancer cell lines (for example, astrocytoma, melanoma, andneuroblastoma), as well as in hematological malignancies such asmultiple myeloma and some B-cell non-Hodgkin lymphomas, constitutingmore than 50% of all cancers in humans.

In one embodiment, the synthetic peptide EPPT1, also known as alpha-M2peptide (YCAREPPTRTFAYWG—SEQ ID NO: 1), derived from the CDR3 Vh regionof a monoclonal antibody (ASM2) raised against human epithelial cancercells, is used in the polymers/micelles and/or methods of thisinvention.

In one embodiment, the targeting moiety enhances attachment to amolecule, or, in another embodiment, a cell in low abundance, which isof interest. In another embodiment, the targeting moiety enhancesattachment following supply of an energy source. In one embodiment, thetargeting moiety is chemically attached to the polymers via a chemicalcross-linking group, or in another embodiment, forms a stableassociation with the polymer, or, in another embodiment, forms anassociation with the polymer, which readily dissociates followingchanges in solution conditions, such as, for example, salt concentrationor pH.

In one embodiment, the targeting moiety may be an antibody, whichspecifically recognizes a molecule of interest, such as a protein ornucleic acid. In another embodiment, the antibody may specificallyrecognize a reporter molecule attached to a molecule of interest. Inanother embodiment, the targeting moiety may be an antibody fragment,Protein A, Protein G, biotin, avidin, streptavidin, a metal ion chelate,an enzyme cofactor, or a nucleic acid. In another embodiment, thetargeting moiety may be a receptor, which binds to a cognate ligand ofinterest, or associated with a cell or molecule of interest, or inanother embodiment, the targeting moiety may be a ligand which is usedto attach to a cell via interaction with its cognate receptor.

In one embodiment, the term “immunoconjugate” refers to an antibodybound to a compound. In one embodiment, the conjugation of an antibodyas described, with a polymer or encapsulated within a micelle of thisinvention represents the immunoconjugates comprising the invention. Inanother embodiment, the compound to which the antibody is bound, isconjugated to a polymer or encapsulated within a micelle of thisinvention, and is to be considered as part of this invention, or inanother embodiment, the antibody, to which a compound is bound, isfurther conjugated to a polymer, or encapsulated within a micelle ofthis invention.

In one embodiment, the term “a labeling agent” refers to a moleculewhich renders readily detectable that which is contacted with a labelingagent. IN one embodiment, the labeling agent is a marker polypeptide.The marker polypeptide may comprise, for example, green fluorescentprotein (GFP), DS-Red (red fluorescent protein), secreted alkalinephosphatase (SEAP), beta-galactosidase, luciferase, or any number ofother reporter proteins known to one skilled in the art. In anotherembodiment, the labeling agent may be conjugated to another moleculewhich provides greater specificity for the target to be labeled. Forexample, and in one embodiment, the labeling agent is a fluorochromeconjugated to an antibody which specifically binds to a given targetmolecule, or in another embodiment, which specifically binds anotherantibody bound to a target molecule, such as will be readily appreciatedby one skilled in the art.

In one embodiment, the polymer may be conjugated to a quantum dot. Inone embodiment, the term “quantum dot” refers to a semiconductornanocrystal with size-dependent optical and electronic properties. Inparticular, the band gap energy of a semiconductor nanocrystal varieswith the diameter of the crystal. “Semiconductor nanocrystal” includes,for example, inorganic crystallites between about 1 nm and about 1000 nmin diameter, or in one embodiment, between about 2 nm and about 50 nm,or in another embodiment, between about 5 nm to about 20 nm (such asabout 5. 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nm)that includes a “core” of one or more first semiconductor materials, andwhich can be surrounded by a “shell” of a second semiconductor material.A semiconductor nanocrystal core surrounded by a semiconductor shell isreferred to as a “core/shell” semiconductor nanocrystal. The surrounding“shell” material may, in another embodiment, have a bandgap greater thanthe bandgap of the core material and can be chosen so to have an atomicspacing close to that of the “core” substrate. The core and/or the shellcan be a semiconductor material including, but not limited to, those ofthe group II-VI (e.g., ZnS, ZnSe, ZnTe, CdS, CdSe, CdTe, HgS, HgSe,HgTe, MgTe and the like) and III-V (e.g., GaN, GaP, GaAs, GaSb, InN,InP, InAs, InSb, AlAs, AlP, AlSb, AlS, and the like) and IV (e.g., Ge,Si, Pb and the like) materials, and an alloy thereof, or a mixture,including ternary and quaternary mixtures, thereof.

In one embodiment, the term “toxin” refers to a molecule which resultsin toxic effects in cells and/or tissue exposed to the toxin. In oneembodiment, the toxin results in cell death, or in another embodiment,cell damage. In one embodiment, the toxin is a natural product of cells,such as bacterial cells, wherein the toxin is used, in one embodiment,when specifically targeted to disease cells as a means of selective cellkilling of diseased cells. In one embodiment, the toxin may comprise anyknown in the art, such as, for example that produced by cholera,tetanus, or any other appropriate species, as will be appreciated by oneskilled in the art.

In another embodiment, this invention also comprises incorporation ofany toxic substance for therapeutic purpose. In one embodiment, thepolymers/micelles of this invention may incorporate an oligonucleotideencoding a suicide gene, which when in contact with diseased cells ortissue, is expressed within such cells. In one embodiment, the term“suicide gene” refers to a nucleic acid coding for a product, whereinthe product causes cell death by itself or in the presence of othercompounds. A representative example of a suicide gene is one, whichcodes for thymidine kinase of herpes simplex virus. Additional examplesare thymidine kinase of varicella zoster virus and the bacterial genecytosine deaminase, which can convert 5-fluorocytosine to the highlycytotoxic compound 5-fluorouracil.

Suicide genes may produce cytotoxicity by converting a prodrug to aproduct that is cytotoxic. In one embodiment, the term “prodrug” meansany compound that can be converted to a toxic product for cells.Representative examples of such a prodrug is gancyclovir which isconverted in vivo to a toxic compound by HSV-thymidine kinase. Thegancyclovir derivative subsequently is toxic to cells. Otherrepresentative examples of prodrugs include acyclovir, FIAU[1-(2-deoxy-2-fluoro-β-D-arabinofuranosyl)-5-iodouracil],6-methoxypurine arabinoside for VZV-TK, and 5-fluorocytosine forcytosine deaminase.

In another embodiment, the polymers/micelles or compositions of thisinvention may comprise at least one molecule, which in anotherembodiment, is a protein, which is immunogenic.

In one embodiment, the term “immunogenic”, refers to an ability toelicit an immune response. Immune responses that are cell-mediated, orimmune responses that are classically referred to as “humoral”,referring to antibody-mediated responses, or both, may be elicited bythe polymers/micelles or compositions of this invention of the presentinvention.

Polymers/micelles or compositions of this invention may, in oneembodiment, be used for vaccine purposes, as a means of preventinginfection.

In another embodiment, the polymers/micelles or compositions of thisinvention are utilized, to provide an immunogenic protein or polypeptideeliciting a “Th1” response, in a disease where a so-called “Th2” typeresponse has developed, when the development of a so-called “Th1” typeresponse is beneficial to the subject. Introduction of the immunogenicprotein or polypeptide results in a shift toward a Th1 type response.

As used herein, the term “Th2 type response” refers to a pattern ofcytokine expression, elicited by T Helper cells as part of the adaptiveimmune response, which support the development of a robust antibodyresponse. Typically Th2 type responses are beneficial in helminthinfections in a subject, for example. Typically Th2 type responses arerecognized by the production of interleukin-4 or interleukin 10, forexample.

As used herein, the term “Th1 type response” refers to a pattern ofcytokine expression, elicited by T Helper cells as part of the adaptiveimmune response, which support the development of robust cell-mediatedimmunity. Typically Th1 type responses are beneficial in intracellularinfections in a subject, for example. Typically Th1 type responses arerecognized by the production of interleukin-2 or interferon γ, forexample.

In another embodiment, the reverse occurs, where a Th1 type response hasdeveloped, when Th2 type responses provide a more beneficial outcome toa subject, where introduction of the immunogenic protein or polypeptidevia the polymers/micelles or compositions of this invention provides ashift to the more beneficial cytokine profile. One example would be inleprosy, where the polymers/micelles or compositions of the presentinvention express an antigen from M. leprae, where the antigenstimulates a Th1 cytokine shift, resulting in tuberculoid leprosy, asopposed to lepromatous leprosy, a much more severe form of the disease,associated with Th2 type responses.

It is to be understood that any use of the polymers/micelles orcompositions of this invention comprising an immunogenic protein forpurposes of immunizing a subject to prevent disease, and/or amelioratedisease, and/or alter disease progression are to be considered as partof this invention.

Examples of infectious virus to which stimulation of a protective immuneresponse is desirable include: Retroviridae (e.g., humanimmunodeficiency viruses, such as HIV-1 (also referred to as HTLV-III,LAV or HTLV-III/LAV, or HIV-III; and other isolates, such as HIV-LP;Picornaviridae (e.g., polio viruses, hepatitis A virus; enteroviruses,human coxsackie viruses, rhinoviruses, echoviruses); Calciviridae (e.g.,strains that cause gastroenteritis); Togaviridae (e.g., equineencephalitis viruses, rubella viruses); Flaviridae (e.g., dengueviruses, encephalitis viruses, yellow fever viruses); Coronaviridae(e.g., coronaviruses); Rhabdoviridae (e.g., vesicular stomatitisviruses, rabies viruses); Filoviridae (e.g., ebola viruses);Paramyxoviridae (e.g., parainfluenza viruses, mumps virus, measlesvirus, respiratory syncytial virus); Orthomyxoviridae (e.g. influenzaviruses); Bungaviridae (e.g., Hantaan viruses, bunga viruses,phleboviruses and Nairo viruses); Arena viridae (hemorrhagic feverviruses); Reoviridae (erg., reoviruses, orbiviurses and rotaviruses);Birnaviridae; Hepadnaviridae (Hepatitis B virus); Parvoviridae(parvoviruses); Papovaviridae (papilloma viruses, polyoma viruses);Adenoviridae (most adenoviruses); Herpesviridae (herpes simplex virus(HSV) 1 and 2, varicella zoster virus, cytomegalovirus (CMV), herpesviruses'); Poxyiridae (variola viruses, vaccinia viruses, pox viruses);and Iridoviridae (e.g. African swine fever virus); and unclassifiedviruses (e.g., the etiological agents of Spongiform encephalopathies,the agent of delta hepatities (thought to be a defective satellite ofhepatitis B virus), the agents of non-A, non-B hepatitis (class1=internally transmitted; class 2=parenterally transmitted (i.e.,Hepatitis C); Norwalk and related viruses, and astroviruses).

Examples of infectious bacteria to which stimulation of a protectiveimmune response is desirable include: Helicobacter pylori, Borelliaburgdorferi, Legionella pneumophilia, Mycobacteria sps (e.g. M.tuberculosis, M. avium, M. intracellulare, M. kansaii, M. gordonae),Staphylococcus aureus, Neisseria gonorrhoeae, Neisseria meningitidis,Listeria monocytogenes, Streptococcus pyogenes (Group A Streptococcus),Streptococcus agalactiae (Group B Streptococcus), Streptococcus(viridans group), Streptococcus faecalis, Streptococcus bovis,Streptococcus (anaerobic sps.), Streptococcus pneumoniae, pathogenicCampylobacter sp., Enterococcus sp., Chlamydia sp., Haemophilusinfluenzae, Bacillus antracis, corynebacterium diphtheriae,corynebacterium sp., Erysipelothrix rhusiopathiae, Clostridiumperfringers, Clostridium tetani, Enterobacter aerogenes, Klebsiellapneumoniae, Pasturella multocida, Bacteroides sp., Fusobacteriumnucleatum, Streptobacillus moniliformis, Treponema pallidium, Treponemapertenue, Leptospira, Actinomyces israelli and Francisella tularensis.

Examples of infectious fungi to which stimulation of a protective immuneresponse is desirable include: Cryptococcus neoformans, Histoplasmacapsulatum, Coccidioides immitis, Blastomyces dermatitidis, Chlamydiatrachomatis, Candida albicans. Other infectious organisms (i.e.,protists) include: Plasmodium sp., Leishmania sp., Schistosoma sp. andToxoplasma sp.

In another embodiment, the polymers/micelles or compositions of thisinvention comprising an immunogenic protein further comprise additionalimmunomodulating proteins.

Examples of useful immunomodulating proteins include cytokines,chemokines, complement components, immune system accessory and adhesionmolecules and their receptors of human or non-human animal specificity.Useful examples include GM-CSF, IL-2, IL-12, OX40, OX40L (gp34),lymphotactin, CD40, and CD40L. Further useful examples includeinterleukins for example interleukins 1 to 15, interferons alpha, betaor gamma, tumour necrosis factor, granulocyte-macrophage colonystimulating factor (GM-CSF), macrophage colony stimulating factor(M-CSF), granulocyte colony stimulating factor (G-CSF), chemokines suchas neutrophil activating protein (NAP), macrophage chemoattractant andactivating factor (MCAF), RANTES, macrophage inflammatory peptidesMIP-1a and MIP-1b, complement components and their receptors, or anaccessory molecule such as B7.1, B7.2, TRAP, ICAM-1, 2 or 3 and cytokinereceptors. OX40 and OX40-ligand (gp34) are further useful examples ofimmunomodulatory proteins.

In another embodiment, the immunomodulatory proteins may be of human ornon-human animal specificity, and may comprise extracellular domainsand/or other fragments with comparable binding activity to the naturallyoccurring proteins. Immunomodulatory proteins may, in anotherembodiment, comprise mutated versions of the embodiments listed, orcomprise fusion proteins with polypeptide sequences, such asimmunoglobulin heavy chain constant domains. Multiple immunomodulatoryproteins may be incorporated within a single construct, and as such,represents an additional embodiment of the invention.

It is to be understood that the polymers/micelles or compositions ofthis invention may comprise multiple immunogenic proteins. In oneembodiment, the immunogenic proteins or peptides are derived from thesame or related species. Vaccine incorporation of multiple antigens hasbeen shown to provide enhanced immunogenicity.

The polymers/micelles or compositions of this invention comprising animmunogenic protein or peptide fragment may generate immune responses ofa variety of types that can be stimulated thus, including responsesagainst the protein or peptide itself, other antigens that are nowimmunogenic via a “by-stander” effect, against host antigens, andothers, and represent additional embodiments of the invention. It isenvisioned that methods of the present invention can be used to preventor treat bacterial, viral, parasitic or other disease states, includingtumors, in a subject.

Combination vaccines have been shown to provide enhanced immunogenicityand protection, and, as such, in another embodiment, the immunogenicproteins or peptides are derived from different species.

In one embodiment, the incorporated groups described herein, which areto comprise the micelles, polymers and/or compositions of thisinvention, may be conjugated to the polymer, or in another embodiment,encapsulated within.

In another embodiment, this invention provides a composition or amicelle comprising a polymer of this invention.

This invention provides amphiphilic polymers, which in one embodiment,are terpolymers. In one embodiment, amphiphilic polymers allow for theformation of spherical nanoparticles, which, in another embodiment,self-assemble into nanospheres. The polymers of this invention, in someembodiments, offer a number of advantages as delivery systems, ascompared to other such systems described in the art, as a result of theunique chemical structure of the polymers of this invention.

In one embodiment, the fundamental unit of the polymers of thisinvention comprises a hydrophilic segment, typically polyethylene glycol(PEG) coupled to a multifunctional, hydrophobic linker molecule. In oneembodiment, the PEG ranges in size from 600-4,400 Daltons.

In one embodiment, the multifunctional hydrophobic linker molecule is atrifunctional linker molecule. In one embodiment, the linker is 5-aminodimethylphthalate or 5-hydroxydimethylphthalate. In one embodiment, ahydrophobic side chain is attached to one of the functional groups ofthe linker via an ether, ester, or amide bond, and the side chain isterminated by a hydrogen or by a functional group such as amino,hydroxyl, or carboxyl. This basic unit is, in another embodiment,further polymerized to yield a base polymer with a molecular size of150-200,000 Da.

The polymers of this invention may assume any structural configuration,which will be a function of, in some embodiments, the chemical makeup ofthe polymers, and the environment to which the polymer is exposed. Insome embodiments, the polymers of this invention may assume a particleconfiguration, comprising a core and shell, or in another embodiment, amicelle configuration.

In one embodiment, when the polymer is dissolved in water above thecritical micelle concentration, about 8 to 12 polymeric units may selfassemble into a spherical micelle consisting of a compact core of sidechains covered by linkers with an external corona of deformable PEGloops.

Depending upon chemical composition, the micelles have, in someembodiments, a molecular weight of about 100-200,000 Da and a diameter(twice the radius of gyration) of about 10 to 300 nm.

In other embodiments, additional agents can be encapsulated in the coreby dissolving the polymer and agent in a solvent, evaporating thesolvent, and dissolving the resulting viscous mixture in water, withappropriate choice of the side chain terminal group. According to thisaspect, and in other embodiments of this invention, a wide variety ofcompounds, such as, for example, various drugs or therapeutic agents(such as, for example, aspirin, naproxen, celebrex, inulin, insulin, andothers, as described herein, and as will be known by one skilled in theart) are encapsulated as cargo within the micelles. In one embodiment,incorporation of these compounds may increase micelle size up to 300 nmin diameter.

The micelle structure may be stabilized, in some embodiments, by thewater-soluble PEG at the exterior surface and by hydrophobicinteractions between the side chains and linkers. When agents with ahydrophilic character are to be encapsulated within the micelle, in someembodiments, the side chains are chosen to retain sufficient hydrophobiccharacter, as to keep the micelle intact. The stability of the micellesto intracellular conditions, for example, in lysozymes, can be varied byselection of coupling between linker and side chain to obtain more orless resistance to low pH, esterases, and other enzymes, in otherembodiments.

In other embodiments, the polymers and/or micelles of this invention maycomprise a targeting agent. In one embodiment, the polymers and/ormicelles of this invention may contain a therapeutic agent as described,and additionally comprise a targeting agent, such that the targetingagent serves to deliver the therapeutic agent to a desired location, fortherapeutic applications. In another embodiment, the targeting agentserves for diagnostic and/or imaging purposes, where an agent isdelivered to a particular site, where verification of delivery isdesired. In another embodiment, the targeting agent serves to provide asensitive means of detection of a particular molecule at a particularsite, for example, the targeting agent directs a micelle or polymer ofthis invention to a tissue which expresses a preneoplastic marker, or acancer associated antigen, wherein the molecule which is being detectedis available in low concentration, and in some embodiments, is notdetectable by existing methods in the art.

In some embodiments, the targeting agent may be coupled to a free PEGhydroxyl at an end of a base polymer chain.

In some embodiments, through the use of various PEG lengths, linkers,side chains, and side chain terminal groups, great flexibility inpolymer/micelle chemical composition, size, structure, and function canbe obtained. In some embodiments, such polymers/micelles may beconstructed via multiple-step reaction pathways that involve synthesisof a suitable monomer with a protected functional group prior to thepolymerization step, followed by deprotection. In other embodiments, thesynthesis may be carried out with a chemical/enzymatic/chemo-enzymaticapproach as exemplified and described further herein.

In one embodiment, the polymers/micelles of this invention incorporate aperfluorocarbon. In one embodiment, the perfluorocarbon is a linear,cyclic or branched fluoroalkyl, preferably perfluoroalkyl, radicaloptionally containing one or more oxygen, nitrogen, chlorine,phosphorous, hydrogen and/or sulfur atoms and/or one or more sulfonyl orcarbonyl groups, or a sulfonyl or carbonyl-containing fluoropolymericgroup.

In one embodiment, the perfluorocarbon may be derived from at least onefluorine-containing polymerizable monomer such as vinyl fluoride,hexafluoropropylene, vinylidene fluoride, trifluoroethylene,trifluorostyrene, chlorotrifluoroethylene, perfluoro(alkyl vinyl ether),tetrafluoroethylene, or cyclic monomers such as —CF═C(OCF₃)O(CF₂)₂₀— or—CF═CFOC(CF₂)₂₀— or mixtures thereof.

In another embodiment, sulfonyl fluoride containing monomers are used,and may include, inter-alia, CF₂═CFOCF₂CF₂SO₂F, CF₂═CFOCF₂CFOCF₂CF₂SO₂F,CF₂═CFOCF₂CFOCF₂CFOCF₂CF₂SO₂F, CF₂═CFCF₂CF₂SO₂F, orCF₂═CFOCF₂CFOCF₂CF₂SO₂F. In other embodiments, fluorocarbon polymerprecursors may comprise polymers containing one or more monomers lackingsulfonyl or carbonyl halide functional groups, but which can be modifiedto include sulfonyl or carbonyl halide groups before or after formingthe polymer. Suitable monomers for such use may includetrifluorostyrene, trifluorostyrenesulfonic acid or the like.

In one embodiment, fluorocarbon polymer precursors having pendantcarbonyl-based functional groups can be prepared in any suitableconventional manner such as in accordance with U.S. Pat. No. 4,151,052or Japanese patent application No. 52 (1977)38486, which areincorporated herein by reference or polymerized from a carbonylfunctional group containing a monomer derived from a sulfonyl groupcontaining monomer by a method such as is shown in U.S. Pat. No.4,151,051 which is incorporated herein by reference. Once prepared, suchpolymers may then be utilized to form the polymers of this invention, aswill be appreciated by one skilled in the art.

A sulfonic acid form of the fluorocarbon polymer precursor can beconverted to the sulfonyl or carbonyl halide form of the fluorocarbonpolymer precursor by a process, such as described, for example, in U.S.Pat. No. 4,209,367 which is incorporated herein by reference

Reaction of the fluorocarbon polymer precursors with amide orsulfonamide-containing reactants or salt thereof can be carried out withthe fluorocarbon polymer precursor being in solid form, solvent-swollenform or in solution with the appropriate reactants in the solid, liquidor gas phase. When the fluorocarbon polymer precursor is in the solidform, the reaction is carried out under anhydrous conditions bycontacting it with the substituted or unsubstituted amide orsulfonamide-containing reactant or salt thereof in a solvent that isnon-reactive with the starting reactants. Representative suitablesolvents include anhydrous polar aprotic solvents such as acetonitrile,tetrahydrofuran, dioxane, or the like, halogenated solvents such aschloroform, or the like. The reaction is carried out in the presence ofan organic non-nucleophilic base in order to scavenge thehalide-containing byproduct of the reaction. Representative suitablenon-nucleophilic bases include alkylamines such as triethylamine,trimethylamine, or the like, pyridines, alkyl pyridines, alkylpiperidines, N-alkyl pyrrolidines, or the like, The reaction can becarried out in the absence of a solvent under conditions where there isenough mobility of the reactants to interact with each other such aswhen the non-nucleophilic base functions as a medium for the reaction.Other suitable halide-containing byproduct scavengers include KF,Na₂CO₃, Zn powder, CsF, or the like. Reaction is effected underanhydrous conditions such as under an inert atmosphere such as argon,nitrogen or the like in a vessel or a glove box at a temperature betweenabout 0 and about 200° C., or in another embodiment, between about 25and about 125° C. Reaction times may be, in other embodiments, betweenabout 5 minutes and about 72 hours, in some embodiments, between about 1hour and about 24 hours. The reaction can be effected while mixing.

When the fluorocarbon polymer precursor is in solution, it is contactedwith the substituted or unsubstituted amide or sulfonamide-containingreactant or salt thereof under the conditions set forth above. Theproduct is recovered as a solid such as by precipitation or by removingthe solvent. Representative suitable solvents for the fluorocarbonpolymer precursor include halogenated solvents such aspolychlorotrifluoroethylene, for example Halocarbon oil,perfluoroalkylamines, for example Fluorinert FC-70, or the like.

In one embodiment, the perfluorocarbon comprises ¹⁹F. In one embodiment,polymers comprising ¹⁹F are particularly useful in applications of thisinvention in imaging and diagnostics, and offer several advantages overtraditionally used agents in such applications, in particular inmagnetic resonance imaging (MRI). ¹⁹F is a magnetically active nucleuswith a relative intrinsic sensitivity 83% of ¹H. The normalconcentration of MRI-observable fluorine in tissue is extremely low.Most tissue fluorine is concentrated in bone mineral as ionic fluorideand therefore exhibits an NMR signal with solid state (broad line)characteristics (extremely short T2) that does not contribute to theimage brightness using conventional MRI techniques. As a consequence,use of polymers/micelles of this invention, comprising ¹⁹F in MRI willresult in a contrast-to-noise ratio that is very high as compared to thegray-scale images typical of ¹H-MRI, with the quality of ¹⁹F-MRI limitedonly by the signal-to-noise ratio of the acquired image.

Another useful property of ¹⁹F for MRI imaging is the linearrelationship between the ¹⁹F spin-lattice relaxation rate (R₁=1/T₁) andlocal oxygen partial pressure, which provides a means for non-invasivepO₂ measurement using ¹⁹F-MRI. The increasing R₁ with increasing pO2also leads to an increase in pixel brightness in T₁-weighted ¹⁹F-MRimages. This property may be exploited in various applications using thepolymers/micelles of this invention, such as, for example, in assessingtumor growth and development (see, for example, Song, Y., et al., NIRspectoscopy. In: Dunn and Swartz (eds.), Oxygen Transport to TissueXXIV, pp. 225-236: Kluwer Academic/Plenum Publishers, 2003), inevaluating respiratory function (see, for example, Thomas, S. R., et al.Investigative Radiology, 32: 29-38, 1997), in ventilation (see, forexample, Laukemper-Ostendorf, S., et al. Magnetic Resonance in Medicine,47: 82-89, 2002), and other applications (see, for example, Noth, U., etal., Magnetic Resonance in Medicine, 42: 1039-1047, 1999; Williams, S.N. O., et al., Biotechnology and Bioengineering, 56: 56-61, 1997). Inanother embodiment, the polymers/micelles of this invention may furtherfind application in cancer imaging, wherein a subject may breatheoxygen-enriched air during ¹⁹F-MRI imaging of theperfluorocarbon-containing polymers/micelles of this invention, whereincreased O₂ inspiration leads to a local pO₂ enhancement, or findapplication in measuring gastric emptying and gastrointestinal transittime in by gavage, and/or imaging pulmonary pathways with fluorinatedgases.

In other embodiments, the polymers/micelles of this invention andcompositions comprising the same may find application in ¹⁹F-MRspectroscopy (MRS), imaging (MRI), and spectroscopic imaging (MRSI) forin vivo quantitative metabolic mapping, as a tool for pharmacokineticstudies, such as, for example, uptake with the chemotherapeutic agent5-fluorouracil and the selective serotonin reuptake inhibitorsfluvoxamine and fluoxetine and their metabolites.

In one embodiment, ¹⁹F MRI may have a conservative detection limit ofabout 20 μM with a 3T magnet (assuming a linear variation of signal tonoise ratio with field strength and inversely with coil diameter),dropping to about 10 μM in a 7T magnet. Moreover, it is expected thatthe very short ¹⁹F T1 of 140 ms (which increases the signal to noiseratio achievable in a given scanning time) reported by Kimura, et al.(Magnetic Resonance Imaging, 22: 855-860, 2004) will not occur in vivo,in using the polymers/micelles of this invention. Further, in oneembodiment of this invention, additional loading of ¹⁹F may beaccomplished using the micelles and compositions of this invention,enhancing the signal.

In another embodiment, the sensitivity of MRI detection of the ¹⁹Fcontaining polymers/micelles of this invention can potentially beincreased several fold by other approaches. The large chemical shifts offluorine generally result in perfluorocarbons having complex chemicalshift spectra, yielding groups of widely separated resonancescorresponding to the different chemical environments of fluorine inthese molecules. Within the chemical shift bands, there are resolved orunresolved isotropic homonuclear J-coupling patterns. ¹⁹F images may beplagued with multiple, at times overlapping, ghost images that resultfrom the convolution of the ideal images with the chemical shiftspectra. The phase modulation due to the J-coupling, which is notrefocused by 180 degree RF pulses, creates additional artifacts.Typically, this situation is addressed, by using chemical shiftselective pulses to image only one resonance band, thereby wastefullydiscarding the bulk of the potentially usable fluorine signal.

In one embodiment, a means of overcoming the chemical shift artifact isto use weak imaging gradients such that the projections of differentchemical shift lines do not overlap; the separate projections may thenbe combined to form a single image of full signal to noise ratio. In oneembodiment, this technique is useful with very high field magnets wherethe chemical shift frequency differences are very large, or in anotherembodiment, in situations where the sensitivity is low and thereforeweak gradients and low spatial resolution (which preserve the signal tonoise ratio) are needed.

In another embodiment, a means of overcoming the chemical shift artifactis via deconvolving the chemical shift spectrum from raw image data, asdescribed (Busse, L. J., et al. Medical Physics, 13: 518-524, 1986).

Advantages of optical imaging methods, as described herein, include theuse of non-ionizing low energy radiation, high sensitivity with thepossibility of detecting micron-sized objects, continuous dataacquisition, and others. At the near infrared region between 700 and 900nm, absorption by intrinsic photoactive biomolecules is low and allowslight to penetrate several centimeters into the tissue. Moreover,imaging in the near-infrared (NIR) region has minimal tissueautofluorescence, which dramatically improves the target/backgroundratio. Optical imaging can be carried out at different resolutions anddepth penetrations. Fluorescence-mediated tomography (FMT) canthree-dimensionally localize and quantify fluorescent probes in deeptissues at high sensitivity, and NIR fluorochromes may be coupled toaffinity molecules, which may serve, in other embodiments, as targetingagents (see, for example, Becker, A., et al. Nature Biotechnology, 19:327-331, 2001; Folli, S., et al. Cancer Research, 54: 2643-2649, 1994).

In another embodiment, the polymers/micelles of this invention allow forthe combination of different imaging modalities.

In another embodiment, the polymers, micelles, compositions, orcombinations thereof of this invention may comprise halogens, asdescribed herein, such as, for example, fluorine or iodine. In oneembodiment, any isotope of the halogen may be used in the polymers,micelles, compositions, or combinations thereof of this invention, andaccording to the methods of this invention, and may find application invarious imaging means, which make use of specific isotopes, as will beappreciated by one skilled in the art.

In one embodiment, this invention provides for the combination of twoimaging modalities which enable MR imaging using ¹⁹F or iron oxide, forexample, as a contrast agent and a fluorescent label, such as the Cy5.5dye as a near-infrared fluorescent (NIRF) probe. Cy5.5 can be coupled toone functional group on the trifunctional linking molecule in place of aside chain. Combined MR/optical probes may be used, in some embodiments,for imaging enzymatic activity, such as for example, protease activityas described (Josephson, L., et al. Bioconjugate Chemistry, 13: 554-560,2002; Kircher, M., et al. Molecular Imaging, 1: 89-95, 2002). In someembodiments, such combination polymers/micelles enable specificrecognition of a desired tissue, for example, produce a high resolutionsignal on MR images, and allow for real-time continuous data acquisitionby NIRF imaging.

In another embodiment, the polymers are synthesized enzymatically. Inone embodiment, the enzymes used to synthesize the polymers or micellesof this invention comprise lipases, such as, for example Candidaantarctica lipase, or in another embodiment, lipase A, or in anotherembodiment, lipase B. In another embodiment, the enzyme may comprise anesterase, or in another embodiment, a protease, such as, for examplepapain or chymotrypsin. In one embodiment, molecular weight of thehydrophilic units is chosen such that its ability to affectpolymerization is considered. In one embodiment, the polymer isfunctionalized with for example, an alkyl group of varying chain length,comprising a polar functionality at the end of the chain.

Polymers obtained by methods as described herein can be characterized bymethods well known in the art. For example, the molecular weight andmolecular weight distributions can be determined by gel permeationchromatography (GPC), matrix assisted laser desorption ionization(MALDI), and static or dynamic light scattering. Physical and thermalproperties of the polymer products can be evaluated by thermalgravemetric analysis (TGA), differential scanning calorimetry (DSC), orsurface tensiometer; the chemical structures of the polymers can bedetermined by, e.g., NMR (1H, 13C NMR, 1H-1H correlation, or 1H-13Ccorrelation), IR, UV, Gas Chromatography-Electron Impact MassSpectroscopy (GC-EIMS), EIMS, or Liquid Chromatography Mass Spectroscopy(LCMS).

In another embodiment, incorporation of perfluorocarbons within thepolymers, micelles and compositions of this invention allows for thefollowing advantages, in applications of ¹⁹F-MRI imaging: such usefacilitates much higher signal-to-noise ratio and greater sensitivitycompared to protons because of the absence of 19F background signals;fluorine is prepared at high concentration in the form of aperfluorocarbon contained within a unique self-assembling polymericmicelle that is small enough to be taken up by cells; and if the micelleexterior is functionalized with a ligand that binds to a receptor foundon most solid tumors but not on normal cells, the resultingreceptor-mediated endocytosis greatly enhances selectivity for tumortissue.

The structure of an embodiment of this invention, a self-assembling,alternating copolymer micelle, is shown schematically in FIG. 1. Eachpolymer, in this embodiment, consists of a hydrophilic polyethyleneglycol (PEG) segment (molecular weight main chain 60-10,000) bound to alinker (aromatic or peptide bond) to which a hydrophobic side chain isbound (via ether or ester linkages) that is terminated by a hydrophobicor hydrophilic group. When dissolved in water above the critical micelleconcentration, about 8 to 12 polymeric units self assemble into aspherical micelle consisting of a compact core surrounded by an outerenvelope of PEG loops that provide biocompatibility. The micelles have amolecular weight of about 100-200,000 and a hydraulic radius rangingfrom about 10 to 30 nm. Additional agents can be encapsulated in thecore. These micelles can be taken up intact by cells, as demonstrated bywith micelles fluorescently labeled on the main chain and on the cargo.Micelle synthesis in which the side chain is a perfluorocarbon wasaccomplished, using perfluoroctyl bromide, and micelles were formedhaving a 30 nm radius, and containing 28% (w/v) fluorine. Additionalperfluorocarbon cargo can be encapsulated inside each micelle,substantially increasing the fluorine content. Only 10⁵ of thesemicelles are estimated to be needed in a cell to achieve a concentrationon the order of 1 mM, which is the minimum required for effective ¹⁹Fimaging. It has been shown that intravenously administeredperfluorocarbon emulsions with diameters 3 to 4 times largerpreferentially accumulate in the interstitial space of solid tumors andcan be detected using ¹⁹F NMR spectroscopy and imaging.

In another embodiment, the polymers form micelles or nanoparticles,which range in size from 5-1000 nm. In one embodiment, the size range isfrom 25-200 nm. In one embodiment, the size range is from 30-200 nm, orin another embodiment, the size range is from 35-200 nm, or in anotherembodiment, the size range is from 40-200 nm, or in another embodiment,the size range is from 45-200 nm, or in another embodiment, the sizerange is from 50-200 nm, or in another embodiment, the size range isfrom 75-200 nm, or in another embodiment, the size range is from 100-200nm, or in another embodiment, the size range is from 125-200 nm, or inanother embodiment, the size range is from 150-200 nm, or in anotherembodiment, the size range is from 175-200 nm, or in another embodiment,the size range is from 35-75 nm, or in another embodiment, the sizerange is from 50-100 nm, or in another embodiment, the size range isfrom 75-200 nm, or in another embodiment, the size range is from 75-150nm, or in another embodiment, the size range is from 50-125 nm, or inanother embodiment, the size range is from 20-100 nm, or in anotherembodiment, the size range is from 20-125 nm.

In another embodiment, the hydrophilic polymer molecular weight may bevaried. In one embodiment, the molecular weight of the hydrophilicpolymer may range from 150-200,000 Da.

In one embodiment, the compositions of this invention, which comprise apolymer and/or micelle of this invention is biocompatible, and inanother embodiment, may comprise pharmaceutically acceptable carriers orexcipients, such as disclosed in Remington's Pharmaceutical Sciences,Mack Publishing Company, Easton, Pa., USA, 1985. The polymers, micellesand/or compositions of this invention may be used in the treatment ordiagnosis of certain conditions such as in tagging, detecting and/orremoving cancer cells for example from a sample or tissue.

In another embodiment, this invention provides a process for producingan amphiphilic polymer comprising perfluorocarbons, the processcomprising the steps of:

contacting a dialkyl 5-hydroxy-isophthalate, a dialkyl5-alkoxy-isophthalate, a dialkyl 5-amino-isophthalate, any derivativethereof or any combination thereof with a polyethylene glycol to form anamphiphilic copolymer; and

linking a perfluorocarbon to said amphiphilic copolymer, thereby being aprocess for producing amphiphilic polymers comprising perfluorocarbons.

In one embodiment, a chemo-enzymatic approach for the synthesis is used.In one embodiment, the processes of this invention may further comprisethe step of protecting the amino group of dialkyl 5-amino-isophthalatewith an amino protecting group.

The phrase “protecting group” as used herein means temporarymodifications of a potentially reactive functional group which protectit from undesired chemical transformations. Examples of such protectinggroups include esters of carboxylic acids, silyl ethers of alcohols, andacetals and ketals of aldehydes and ketones, respectively. The field ofprotecting group chemistry has been reviewed (Greene, T. W.; Wuts, P. G.M. Protective Groups in Organic Synthesis, 2nd ed.; Wiley: New York,1991).

In another embodiment, the processes of this invention may furthercomprise the step of protecting the hydroxy group of a dialkyl5-hydroxy-isophthalate with an hydroxy protecting group.

In one embodiment, enzymatic polymerization of a hydrophilic with amultifunctional linking molecule to form the copolymer backbone isconducted initially. In one embodiment, the linking moiety is dissolvedin the hydrophillic liquid without any additional solvent, enzyme isadded, and polymerization is carried out at high temperature, forexample at about 90° C., under vacuum.

In one embodiment, the process of this invention comprises synthesis ofa polymer comprising a perfluorocarbon, wherein the perfluorocarbon islinked to a hydroxyl group, amino group, or combination thereof of theisophthalate, and in one embodiment, the attachment of perfluorocarbonto isophthalate is via an enteric bond, an amide bond, or a combinationthereof.

In one embodiment, the synthesis of the basic polymer takes place in twosteps, which comprise, inter-alia, attachment of a targeting agentand/or labeling agent subsequent to the basic polymer formation.

In one embodiment, the hydrophilic moiety is a PEG oligomer (n=10-34)and the multifunctional linker is dimethyl 5-hydroxyisophthalate.

According to this aspect, and in one embodiment, the reaction is atrans-esterification, and the methanol formed during the reaction isremoved under vacuum. In one embodiment, the method employs the use oflipase B from Candida antartica, and takes advantage of theregioselectivity of the enzyme, such that the phenolic group does nottake part in the polymerization, thereby giving a polymer with areactive functional group. This reactive functional group, in turn, maybe used for further chemical reactions, in this case attachment of ahydrophobic group with either an ether or ester linkage using standardgroup replacement chemistry, as will be appreciated by one skilled inthe art.

In one embodiment, the enzyme may be immobilized within porouspoly(methyl methacrylate) beads, such as, for example, that available asNovozyme 435 from Novozyme A/S).

In other embodiments, any number of multifunctional linkers may be used,such as, for example, those with hydroxy or amino functional group and avariety of hydrophobic moieties may be attached with and without anadditional terminal functional group. For example, and in otherembodiments, polymers comprising linkers such as dimethyl 5-aminoisophthalate, amino malonic acid, aspartatic and glutamic acid aslinkers, have been attached to hydrocarbon chains which have afunctionality of hydroxy, carboxy, amino, or guanidinyl groups at theend of the chain, by methods well known in the art [see for example,Kumar, R., et al. Journal of the American Chemical Society, 126:10640-10644, 2004; Kumar, R., et al., Green Chemistry, 6: 516-520, 2004;Kumar, R., et al., Journal of Macromolecular Science: Pure and AppliedChemistry, A40: 1283, 2003; Tyagi, R., et al., Polymer Preprint, 44:778, 2003; Sharma, S. K., et al. Polymer Preprint, 44: 791, 2003;Sharma, S. K., et al., Journal of Macromolecular Science: Pure andApplied Chemistry, A41: 1459, 2004, all of which are incorporated hereinby reference].

In one embodiment, the linkage at the aromatic oxygen may be an ester orether linkage. For example, if an aminophthalate is used, the connectionof the side chain is an amide link.

In other embodiments, the length of the PEG or hydrophilic segment maybe varied over a wide range, such as disclosed, for example in Kumar,R., et al. Journal of Macromolecular Science, A39: 1137-1149, 2002, suchthat the hydrophilicity/hydrophobicity ratio for the polymer may becontrolled. In other embodiments, the polymerization conditions may becontrolled, such that a structure is obtained in which hydroxyl groupsof the PEG component are available on both ends of the polymer chain,which in other embodiments, may be used for subsequent chemicalmodification. These hydroxyl groups may be used, in other embodiments,to attach a targeting agent, such as for example, the peptide EPPT1, asexemplified herein, or, in another embodiment, a labeling agent, suchas, for example, a fluorescent compound.

The polymers may form micelles, when in solution. Characterization ofthe polymers in aqueous solution with light scattering techniques,demonstrates formation of nanoparticles via a self-assembly process witha PEG external surface and a hydrophobic internal cavity. The ratio ofthe radius of gyration, Rg, (static light scattering) to thehydrodynamic radius, Rh, (dynamic light scattering) is about 1.75,indicating that the nanoparticles correspond to a hollow spheroidalstructure. Attachment of functional groups at the end of the hydrophobicchains allows modification of the cavity of these nanospheres, whichaffects their size and stability as well as the nature of cargo that canbe encapsulated. Static light Scattering of the nanospheres gives Rg inthe range of 10-80 nm. The size of the nanospheres and their stabilityare influenced by the length of the PEG oligomers and the nature of thehydrophobic group. The incorporation of hydrophobic side chains may addto the stability of the micelles.

In one embodiment, the polymers will have a molecular weight of around200,000 Da and contain 10-12 copolymer chains per nanosphere, each about20,000 Da in molecular weight.

A wide variety of small molecules including drugs may be encapsulatedwithin the micelles of this invention. Larger molecules such as proteins(insulin) and polysaccharides (inulin) have also been encapsulated sincethe nanospheres may adjust to the size of the encapsulant molecule inthe self assembly process. In order to encapsulate smaller molecules, aprotocol as described (Kumar, R., et al., Journal of the AmericanChemical Society, 126: 10640-10644, 2004; Sharma, S. K., et al.,Chemical Communication 23: 2689-2691 2004) may be used. The polymer andcargo are dissolved together in an organic solvent, such as chloroform,and then the solvent is evaporated to dryness. The residue is dissolvedin water and any unencapsulated material removed by filtration. Theaqueous solution is freeze-dried, kept until needed, and thenreconstituted with water to give clear solutions of the encapsulatedmaterial. The amount of encapsulant as a fraction by weight may bedetermined by several methods. When the UV absorptivity of theencapsulant is sufficiently different from the polymer, UV spectroscopymay be used. In other cases, ¹H-NMR may be used, as described in Sharma,supra. Typically, a ratio of 1:4 or 1:5 cargo to polymer weight ratiosare used. As the ratio increases, the fractional mass of the cargoincreases, and the nanoparticle size increases until a maximum isreached.

The choice of starting reagents used to construct the polymers of thisinvention may be tailored, for example, for the attachment of differenttypes of pendant groups, for example to a hydroxyl group, includingalkyl or alkenyl chains, aryl groups, carboxyl-containing groups, aminogroups, ammonium groups, and/or additional hydroxyl groups. In anotherembodiment, appropriate choice of the pendant group functionalities,enables enhanced polymer interaction with incorporated molecules, suchas therapeutic compounds, fluorochromes, perfluorocarbons, etc., foroptimal conjugation of the various functional groups herein described.

For example, a carboxyl-containing functional pendant group can interactwith nitrogen bases (e.g., primary, secondary, or heterocyclic amines),and can form Schiff bases under appropriate conditions. By choosingappropriate encapsulation conditions, the resulting structure can beformed in such a way that the drug is well held in the core of themicelle, protected from the physiological milieu. As another example, acarboxylic acid group on the drug can be ion-paired with a pendant amine(e.g., a secondary or tertiary amine). The resulting ion pair can beformed in such a fashion that it resides substantially within the coreof the micelle. Such pendant groups can be incorporated into the polymerwith relative ease, using well-known synthesis methods. Thus, thepolymers can be readily tailored to create vehicles that meet thespecific requirements of a given guest drug molecule, for example, andin one embodiment, or any other molecule for delivery, using thepolymers and/or micelles of this invention.

Synthesis of Fluorine-Containing Nanoparticles

Fluorine incorporation into the base copolymer may be via any number ofstandard methods of formation of ester or ether linkages to attach aperfluorinated chain. For example, and in one embodiment of thisinvention, the amphiphilic copolymer (with PEG, n=15) is mixed withperfluoro octanoyl chloride under basic conditions to attach an acylperfluoro group to a phenolic moiety as the hydrophobic side chain. Theattachment may be confirmed with IR spectroscopy and ¹⁹F-NMR. Afluorine-modified polymer thus formed demonstrated nanoparticles with anRg of about 75 nm, as determined by static light scattering. Itcontained 28% (w/w) fluorine, corresponding to about 3,800 ¹⁹F atoms pernanoparticle.

The amphiphilic copolymers with perfluorocarbon side chains were thenused to further encapsulate 1,1,2,2,-tetrahydro perfluorododecanol (20%w/w) using the same procedure as described above. The amount ofperfluorocarbon cargo encapsulated by the fluorinated polymer wasdetermined by integration of fluorine NMR spectra. The entire particlecontained 42% (w/w) fluorine, corresponding to almost 6,000 ¹⁹F atomsper nanoparticle. Loading may be increased by at least a factor of twoto 12,000 ¹⁹F atoms per nanoparticle. Assuming a cell volume of 10³ μm³,uptake of 10⁵, 10⁶, or 10⁷ of the nanoparticles per cell is obtainable,and result in cellular fluorine concentrations of about 2, 20, or 200mM, respectively, amounts sufficient for efficient imaging, in clinicalsettings.

The physical and chemical properties of the polymers/microspheres ofthis invention may readily be determined with standard techniques suchas IR spectroscopy, NMR spectroscopy, gel permeation chromatography, andlight scattering (dynamic and static).

In one embodiment, the targeting agent is a peptide, which in oneembodiment binds to an underglycosylated mucin-1 protein, which in oneembodiment is EPPT1, as described herein. In one embodiment, the EPPT1peptide is based on the CDR 3 VH and framework regions of the idiotypeof a murine antitumor monoclonal antibody ASM2 directed against thepolymorphic epithelial human mucin epitope (Hussain, R., et al.Peptides: Chemistry, Structure, and Biology. Proceedings of the 14thAmerican Peptide Symposium, England, 1996, pp. 808-809).

In one embodiment, synthesis of the polymer comprising the EPPT1 peptidewill comprise polymerization with two linkers, one of which will be in asmall amount (1-5%) to give the polymer as shown in FIG. 6. Thesynthesis is conducted such that PEG hydroxy groups are at the ends ofthe chain, and perfluorocarbon side chains are attached to the linkerhydroxyls by standard acylation procedures to form the ester linkagewith the polymer backbone. Numerous fluorine-containing polymers may beprepared via this route, including the formation of (CF₂)₈CF₃,(CF₂)₆CF₃, (CF₂)₃CF₃, CH₂OCH₂(CF₂)₈CF₃, CH₂OCH₂(CF₂)₆CF₃,CH₂OCH₂(CF₂)₄CF₃ or CH₂OCH₂CH₂(CF₂)₁₁CF₃. In other embodiments, thesynthetic processes of this invention are highly flexible, enabling thevariation of the chain length (from 5 to 13 carbons) and the relativenumber of fluorine atoms to alter the hydrophobicity of the side chain.

The effect of any of the parameters on polymer/micelle-loading andstability may be evaluated, by any number of methods known to oneskilled in the art, and a number of encapsulating materials may beevaluated concurrently, including perfluorodecalin,bromo-perfluoroheptane, and perfluoro-crown ether. Cy5.5 may be attachedto the polymers to enable NIRF determination, EPPT1 peptides fortargeting, and radioiodine for cell binding and biodistribution studies.

In one embodiment, the process of this invention comprises linking aperfluorocarbon to the amphiphilic copolymer via, inter-alia, convertingthe amino group (—NH2) of the isophthalate to —NH—R1, wherein R1 is asdefined herein.

In one embodiment, the process of this invention comprises linking aperfluorocarbon to an amphiphilic copolymer via, inter-alia, alkylatingthe hydroxy group (—OH) of the isophthalate to produce —(CH₂)_(q)CO—R₂,wherein R₂ is as defined herein.

In another embodiment, this invention provides a polymer or micelle, orcomposition comprising a product of a process of this invention.

In another embodiment, this invention provides a method of imaging acell, the method comprising the steps of contacting a cell with anamphiphilic polymer of this invention and imaging said cell, wherebysaid polymer enables the imaging of said cell.

Attachment of Fluorescent Probe

Fluorochromes may readily be attached to a polymer of this invention,and represent an embodiment thereof. As exemplified herein, Rhodamine Bwas converted to its acid chloride using oxalyl chloride. Treatment ofthe polymer (substituted with a decane chain as the hydrophobic group)with the acid chloride and base formed an ester linkage with the CH₂OHgroups at the ends of the polymer chains, binding it covalently to thepolymer, and attachment did not interfere with nanosphere formation asdetermined by light scattering.

Neuroblastoma cells incubated with nanospheres with Rhodamine B attachedto the polymer, and brilliant green loaded within the spheres, showednanosphere polymer and cargo penetrated cells, as evidenced bycolocalization of the two fluorescent signals, indicating that thenanoparticles entered the cell with its cargo intact.

In one embodiment, a fluorescent molecule is attached to a polymer ofthis invention. In one embodiment, the fluorescent molecule is Cy5.5. Inone embodiment, Cy5.5 is attached to amine groups of the base polymerand non-reacted dye may be removed by any number of conventional means,such as, for example, via column chromatography.

In another embodiment, the fluorescent molecule may be introduced withina targeting moiety which is coupled to a polymer of this invention. Forexample, an EPPT1 peptide (YCAREPPTRTFAYWG-SEQ ID NO: 1) is modified tointroduce a FITC label, to produce a final peptide with the followingsequence: Y-C(ACM)-A-R-E-P-P-T-R-T-F-A-Y-W-G-K(FITC)K (SEQ ID NO: 2).

In one embodiment, peptides of this invention may be purified fromappropriate sources, or in other embodiments, may be synthesized, bymeans well known in the art. In one embodiment, peptides may besynthesized on an automatic synthesizer using Fmoc chemistry with HBTUand HOBT. They may be further purified by C18 reverse phase HPLC.Molecular weight may be determined by MALDI mass spectroscopy.

In one embodiment, both the targeting moiety and the polymer may belabeled with fluorescent markers, or, in another embodiment, any otheragent, as described. In one embodiment, such conjugation may beaccomplished by any number of methods known in the art, such as, forexample, that of Zalipsky, et. al. (Advanced Drug Delivery Reviews, 54:459-476, 2002), or Roberts, M. J. et al. Advanced Drug Delivery Reviews,54: 459-476, 2002).

In one embodiment, polymers or micelles of this invention may beradiolabeled. For example, incorporation of Na ¹²⁵I may be accomplishedusing the Iodogen method (Pierce, Rockford, Ill.) using available Tyrwithin the peptide sequence, in conjugated polymers. In anotherembodiment, the basic polymer backbone may be radiolabeled with the sameprocedure via substitution of the isophthalate ring similar to that ofthe tyrosine aromatic ring, in peptide or protein-conjugated polymers.

In another embodiment, the methods of this invention are directed to theimaging of individual cells, a group of cells, a tissue, an organ or acombination thereof.

In one embodiment, imaging is accomplished with computed tomography,computed radiography, magnetic resonance imaging, fluorescencemicroscopy, angiography, arteriography, or a combination thereof. In oneembodiment, a cell is contacted with a polymer of this invention,ex-vivo, and is subsequently implanted in a subject. In one embodiment,the cell is inter-alia, labeled with a labeling agent as describedherein, and may further comprise a therapeutic compound, and/or inanother embodiment, the therapeutic compound is labeled with a labelingagent, and in one embodiment, the delivery of the cell and/ortherapeutic compound may be verified by imaging the labeling agent.

In one embodiment, the imaging methods of this invention are conductedon a subject. In another embodiment, the imaging methods are conductedon a sample taken from a subject. In one embodiment, the subject has oris suspected of having cancer, or in another embodiment, atheroscleroticlesions, or in another embodiment, is infected, or in anotherembodiment, has ischemica.

In one embodiment, the imaging methods as described herein may comprisenear infrared fluorescence imaging. In one embodiment, an advantages ofsuch optical imaging methods may include the use of non-ionizing lowenergy radiation, high sensitivity with the possibility of detectingmicron-sized objects, continuous data acquisition, and the developmentof potentially cost-effective equipment. Optical imaging can be carriedout at different resolutions and depth penetrations.Fluorescence-mediated tomography (FMT) can three-dimensionally localizeand quantify fluorescent probes in deep tissues at high sensitivity.Several NIR fluorochromes have recently been coupled to affinitymolecules (Becker, A., et al. Nature Biotechnology, 19: 327-331, 2001;Folli, S., et al Cancer Research, 54: 2643-2649, 1994, and can beadapted to comprise the polymers or micelles of this invention, as willbe appreciated by one skilled in the art.

In one embodiment, the imaging methods as described herein may comprisenuclear imaging methods. Nuclear imaging is based on labeling moleculeswith a radioactive atom before their release in the system under study.Since photons of relatively high energy (>80 keV) can escape from thehuman body, it is possible to follow over time the 3D spatialdistribution of the radioactive tracer through detection of the emittedradiation. A large variety of isotopes can be imaged. Their broadestclassification is perhaps that in gamma and positron emitters: theformer family is at the basis of single photon emission methods (such asplanar scintigraphy and tomography, or SPECT), and the latter is used inPositron Emission Tomography (PET). Unlike in MRI or computed tomography(CT), the signal detected in nuclear imaging techniques is theradioactive emission of a single atom. Because these emissions arespecific to the radioisotope used, and because it is possible withstandard physics instrumentation to detect the emission of a singleatom, nuclear imaging enjoys the advantages of both high specificity andsensitivity. Structural information, however, may be obtained only asfar as the radiotracer redistributes following anatomical structures.Resolution of clinical scanners may be limited to about 5-6 mm for PETand ˜1 cm for SPECT, thus, nuclear imaging methods are often used tocomplement the information provided by CT and/or MRI scans in thecontext of multimodality imaging, and may be applied in this mannerherein, representing an embodiment of this invention. In one embodiment,nuclear imaging is used in particular because of its sensitivity toextremely small quantities of matter. For example, it has recently beenestimated that PET can detect as few as a cluster of 250 cells eachbearing 30 Bq of ¹⁸F, which corresponds to 2.1 fg.

While PET techniques achieve good resolution with high sensitivity(2-4%), common positron emitters such as ¹⁸F has a relatively shorthalf-life, which may affect it's widespread applicability. In oneembodiment, however, nanoparticle encapsulation as described herein, maylengthen this half-life and enhance it's applicability.

In another embodiment, different iodine isotopes can be chosen forradioactive labeling of compounds. In one embodiment, ¹²³I, ¹²⁵I and¹³¹I can be used to obtain molecules with the same chemical andbiological characteristics but different imaging and dosimetricproperties. ¹³¹I In one embodiment, the isotope for imaging is ¹²³I (159keV), or in another embodiment, 37 MBq of ¹²³I-MIBG, which results in anexposure to a radiation dose no higher than 1.8 MBq of ¹³¹I-MIBG.

In radioimmunotherapy (RIT), cytotoxic radiation from therapeuticradioisotopes is delivered to tumors via antibodies or peptides thatbind to tumor-specific or tumor-associated antigens (116). Radioactivemetal ions can be attached to an antibody through a metal chelatingagent (117). One advantage for RIT over other immunotherapies, such asimmunotoxins, is that there is no need to target every tumor cell tocause an antitumor effect at the cellular level because nontargetedcells can be irradiated and often killed by radiation from targetedneighboring cells. With immunotoxins, each tumor cell must be targetedfor the antitumor effect to occur at the cellular level (116).

In another embodiment, some of the radioisotopes may serve a dualpurpose, such as, in one embodiment, for imaging the sites to which theradioisotope is delivered, and in another embodiment, as part ofradiotherapy, including radioimmunotherapy. In one embodiment, ¹³¹I and⁹⁰Y are used. ¹³¹I, in one embodiment, may be attached to an antibody orpeptide by simple techniques (such as the IODOGEN or chloramine-Tmethods), and may be imaged by instrumentation which detects γ-emission,while β-emission serves for therapeutic application in the subject.

Delivery of Therapeutic Compounds

The micelles of this invention may be used to encapsulate any number oftherapeutic agents, individually or in combination. Some examples oftherapeutic compounds are described herein, such as, for example,non-steroidal anti-inflammatory drugs such as aspirin and naproxen, orothers as described hereinabove. In one embodiment, the terms “drugs”and “therapeutic compound” are interchangeable, and refer, in someembodiments to compounds producing symptom palliative effects, delay inseverity of symptoms or disease progression, inhibition of disease, orany positive effect attributable to the therapy, or a combinationthereof.

Delivery to a subject through various routes, for example,intravenously, intramuscularly, topically, etc., which may vary, in someembodiments, as a function of the desired site of delivery, or timing,or combination thereof.

Any number of assays may be utilized in order to verify that the drugsare delivered to the appropriate site, and are functional, and suchassays will be tailored for the particular drug utilized As an example,a human cell line such as OM10.1 (Butera et al., AIDS Res. Hum.Retroviruses, 8:991-995, 1992), which is chronically infected withHIV-1, may be used to test antiviral activities of polymer encapsulatedanti-HIV drugs, which is one embodiment of this invention. Such an assaymay be conducted as described, in for example, Critchfield et al., AIDSRes. Hum. Retroviruses, 12:39-46, 1996). Anti-viral effects can bedetermined through a variety of assays, including measuring HIV-1 p24antigen levels, for example, using a commercially available ELISA kit(Coulter), and for reverse transcriptase (RT) activity, using acommercially available chemiluminescent ELISA RT assay such as that soldby Boehringer Mannheim, each according to the manufacturer'sinstructions. Inhibition of viral cell-to-cell spread may be measured,in another embodiment, serving as an indicator of anti-viral efficacy,using a model system, for example, as described (Rabin et al, 1996; Satoet al., 1992).

It is to be understood that any assay for measuring a particularactivity which is modulated by the therapeutic compound may be employed,as a means of determining the efficacy of the compound, in oneembodiment, optimal loading of the compound, in another embodiment,timing and dosage, in another embodiment, or a combination thereof.

Targeting of Specific Agents Using the Polymers and Micelles of thisInvention

FITC-labeled EPPT1 peptide-conjugated micelles were exemplified herein.Any number of cells or cell lines may be incubated with the taggedmolecules and targeting of desired cells and/or uptake may bedemonstrated by conventional means, including microscopy, FACS analysis,western blot analysis, and others.

In vivo imaging can be readily performed on subjects exposed to labeledpolymers/micelles. MR-imaging or NIRF analysis may be used, as well asfluorescence microscopy of excised target tissue, the images of whichmay be compared to those obtained by MIR or NIRF.

In another embodiment, this invention provides a method of targeteddelivery of at least one agent in a subject comprising the steps ofadministering to said subject an amphiphilic polymer of this invention,wherein said polymer comprises said agent and a targeting agent.

In another embodiment, this invention provides a method for detectingneoplastic cells in a subject, comprising contacting a cell in, or acell derived from said subject with an effective tumor-detecting amountof an amphiphilic polymer of this invention, wherein said polymercomprises a targeting moiety specific for neoplastic cells; anddetecting any of said polymer associated with neoplastic cells presentin said subject.

In another embodiment, this invention provides a method of imaging acell, the method comprising the steps of contacting a cell with anamphiphilic polymer of this invention and imaging said cell, wherebysaid polymer enables the imaging of said cell.

In another embodiment, this invention provides a method of targeteddelivery of at least one agent in a subject comprising the steps ofadministering to said subject an amphiphilic polymer of this invention,wherein said polymer comprises said agent and a targeting agent.

In one embodiment, multiple targeting moieties, may be incorporated inthe polymers or micelles of this invention. In one embodiment, multiplesof the same targeting moiety will be incorporated, or in anotherembodiment, multiple targeting moieties, which target the same cell ortissue, may be incorporated.

In another embodiment, this invention provides a method for detectingneoplastic cells in a subject, comprising contacting a cell in, or acell derived from said subject with an effective tumor-detecting amountof an amphiphilic polymer of this invention, wherein said polymercomprises a targeting moiety specific for neoplastic cells; anddetecting any of said polymer associated with neoplastic cells presentin said subject.

As used herein, the term “contacting a target cell” refers to bothdirect and indirect exposure of the target cell to a polymer, micelle orcomposition of this invention. In one embodiment, contacting a cell maycomprise direct injection of the cell through any means well known inthe art, such as microinjection. It is also envisaged, in anotherembodiment, that supply to the cell is indirect, such as via provisionin a culture medium that surrounds the cell.

Protocols for introducing the polymers, micelles or compositions of theinvention to cells and subject may comprise, for example: direct uptaketechniques, injection, receptor-mediated uptake (for further detail see,for example, “Methods in Enzymology” Vol. 1-317, Academic Press, CurrentProtocols in Molecular Biology, Ausubel F. M. et al. (eds.) GreenePublishing Associates, (1989) and in Molecular Cloning: A LaboratoryManual, 2nd Edition, Sambrook et al. Cold Spring Harbor LaboratoryPress, (1989), or other standard laboratory manuals), and others, aswill be appreciated by one skilled in the art. It is to be understoodthat any direct means or indirect means of intracellular access ofpolymers, micelles or compositions of the invention is contemplatedherein, and represents an embodiment thereof.

In one embodiment, the cell which is targeted for uptake of a polymer,micelle or composition of this invention may include any epithelialcell, muscle cell, nerve cell, lung cell, kidney cell, liver cell,astrocyte, glial cell, prostate cell, professional antigen presentingcell, lymphocyte, M cell, or any other cell in the body, where thepolymers or micelles or compositions of this invention may be useful.

In one embodiment, the polymers or micelles or compositions of thisinvention may be administered in any effective, convenient mannerincluding, for instance, administration by intravascular (i.v.),intramuscular (i.m.), intranasal (i.n.), subcutaneous (s.c.), oral,rectal, intravaginal delivery, or by any means in which the polymers ormicelles or compositions of this invention can be delivered to tissue(e.g., needle or catheter). Alternatively, topical administration may bedesired for insertion into epithelial cells. Another method ofadministration is via aspiration or aerosol formulation.

For administration to mammals, and particularly humans, it is expectedthat the physician will determine the actual dosage and duration oftreatment, which will be most suitable for an individual and can varywith the age, weight and response of the particular individual.

According to this aspect of the invention, the disease for which thesubject is thus treated may comprise, but is not limited to: musculardystrophy, cancer, cardiovascular disease, hypertension, infection,renal disease, neurodegenerative disease, such as alzheimer's disease,parkinson's disease, huntington's chorea, Creurtfeld-Jacob disease,autoimmune disease, such as lupus, rheumatoid arthritis, endocarditis,Graves' disease or ALD, respiratory disease such as asthma or cysticfibrosis, bone disease, such as osteoporosis, joint disease, liverdisease, disease of the skin, such as psoriasis or eczema, ophthalmicdisease, otolaryngeal disease, other neurological disease such as Turretsyndrome, schizophrenia, depression, autism, or stoke, or metabolicdisease such as a glycogen storage disease or diabetes. It is to beunderstood that any disease whereby expression of a particular protein,provision of a therapeutic protein, provision of a drug, inhibition ofexpression of a particular protein, etc., which can be accomplished viathe use of the polymers, micelles or compositions of this invention issought, is to be considered as part of this invention.

The following examples are presented in order to more fully illustratesome embodiments of the invention. They should, in no way be construed,however, as limiting the scope of the invention.

EXAMPLES Example 1 Synthesis and Characterization of Novel Multi-ModalMicelle Nanoparticle

A chemo-enzymatic approach (Kumar, R., et al. Journal of MacromolecularScience, A39: 1137-1149, 2002; Kumar, R., et al. Journal of the AmericanChemical Society, 126: 10640-10644, 2004) was developed for the designand synthesis of a complex polymer structure that forms nanospheres,which in one embodiment possesses targeted multi-modal imagingcapability. The polymer self-assembles into spherical nanoparticles.Synthesis of the basic polymer takes place in two steps, followingwhich, in one embodiment of the invention, attachment of a targetingpeptide and fluorescent moiety may occur subsequent to the basic polymerformation.

Enzymatic polymerization of a PEG oligomer (n=10-34) with atrifunctional linking molecule (dimethyl 5-hydroxyisophthalate) isconducted and results in the formation of a copolymer backbone (FIG. 1).The phthalate is dissolved in liquid PEG without any additional solvent,enzyme is added, and the polymerization is carried out at 90° C. undervacuum, as described (Kumar, R., et al. Journal of MacromolecularScience, A39: 1137-1149, 2002; Kumar, R., et al. Journal of the AmericanChemical Society, 126: 10640-10644, 2004). The reaction is atransesterification, with the methanol formed removed under vacuum. Themethod takes advantage of the regioselectivity of the enzyme (lipase Bfrom Candida antartica immobilized within porous poly(methylmethacrylate) beads, available as Novozyme 435 from Novozyme A/S) suchthat the phenolic group does not take part in the polymerization,thereby giving a polymer with a reactive functional group. This reactivefunctional group may be used for further chemical reactions, in thiscase, attachment of a hydrophobic group with either an ether or esterlinkage using standard group replacement chemistry.

The generality of the method enables utilization of a number oftrifunctional linkers with hydroxy or amino groups as the remainingfunctional group and attaching a variety of hydrophobic moieties withand without an additional terminal functional group. Specifically,dimethyl 5-amino isophthalate (Kumar, R., et al. Biocatalytic “Green”synthesis of PEG-based aromatic polyesters: optimization of thesubstrate and reaction conditions. Green Chemistry, 6: 516-520, 2004),amino malonic acid (Kumar, R., et al. Journal of Macromolecular Science:Pure and Applied Chemistry, A40: 1283, 2003), and aspartatic andglutamic acid (Tyagi, R., et al. Polymer Preprint, 44: 778, 2003) havebeen used as linkers, and hydrocarbon chains which have a functionalityof hydroxy (Kumar, R., et al. Journal of the American Chemical Society,126: 10640-10644, 2004), carboxy (Kumar, R., et al. Journal of theAmerican Chemical Society, 126: 10640-10644, 2004), amino (Sharma, S.K., et al. Polymer Preprint, 44: 791, 2003), or guanidinyl (Sharma, S.K., et al. Journal of Macromolecular Science: Pure and AppliedChemistry, A41: 1459, 2004) groups have been attached at the end of thechain. The linkage at the aromatic oxygen may be an ester or ether. Ifthe aminophthalate is used, the connection of the side chain would be anamide link. The length of the PEG or hydrophilic segment may be variedover a wide range (Kumar, R., et al. Journal of Macromolecular Science,A39: 1137-1149, 2002), thus providing control of thehydrophilicity/hydrophobicity ratio as well as functionality. Thepolymerization conditions can be controlled such as to obtain astructure with the hydroxyl group of the PEG component available on bothends of the polymer chain for subsequent chemical modification. Thesehydroxyl groups are used to attach the peptide EPPT1 or to attach afluorescent moiety if the peptide is absent.

Characterization of these polymers in aqueous solution with lightscattering techniques (Chen, M. H., et al. Polymer Preprints, 44:1199-1200, 2003) has shown that they form nanoparticles via aself-assembly process with a PEG external surface and a hydrophobicinternal cavity (FIG. 2). The ratio of the radius of gyration, Rg,(static light scattering) to the hydrodynamic radius, Rh, (dynamic lightscattering) is about 1.75, which indicates that the nanoparticlescorrespond to a spheroidal structure. Attachment of functional groups atthe end of the hydrophobic chains allows modification of the cavity ofthese nanospheres, which affects their size and stability as well as thenature of cargo that can be encapsulated. Static light scattering ofthese nanospheres gives Rg in the range of 10-80 nm. The size of thenanospheres and their stability are influenced by the length of the PEGoligomers and the nature of the hydrophobic group. The nanospheres arequite stable as long as the side chains have significant hydrophobiccharacter. They have a molecular weight around 200,000 Da and contain10-12 copolymer chains per nanosphere, each about 20,000 Da in molecularweight.

Example 2 Encapsulation of Cargo Materials

A wide variety of small molecules including drugs may be encapsulated bythe self-assembly process described in Example 1. Larger molecules suchas proteins (insulin) and polysaccharides (inulin) have beenencapsulated, since the nanospheres may adjust to the size of theencapsulant molecule in the self-assembly process. In order toencapsulate smaller molecules, a simple protocol is used (Kumar, R., etal. Journal of the American Chemical Society, 126: 10640-10644, 2004,Sharma, S. K., et al. Chemical Communication 23: 2689-2691, 2004). Thepolymer and cargo are dissolved together in an organic solvent, such aschloroform, and then the solvent is evaporated to dryness. The residueis dissolved in water and any unencapsulated material removed byfiltration. The aqueous solution is freeze-dried, kept until needed, andthen reconstituted with water to give clear solutions of theencapsulated material. The amount of encapsulant as a fraction by weighthas been determined by several methods. When the UV absorptivity of theencapsulant is sufficiently different from the polymer, UV spectroscopyis used. In other cases, ¹H-NMR (Sharma, S. K., et al., supra) is used.Typically, a ratio of 1:4 or 1:5 cargo to polymer weight ratios has beenused. As the ratio increases, the fractional mass of the cargoincreases, and the nanoparticle size increases until a maximum isreached.

The attachment of targeting peptides was also evaluated. The chemistryof attachment of peptides was straight forward with peptides/proteinsattached at one at the ends of the polymeric chains which, upon choosingthe proper polymerization procedure, have the PEG chain at both ends.Since in one embodiment, nanospheres have 8-10 chains per particle, thisshould give 16-20 peptide units per particle.

A monomethoxy PEG chain was used to simulate the chemistry at the end ofa long PEG chain. The terminal hydroxy group was activated by asuccinimidyl ester according to the method of Miron and Wilchek (Miron,T. and Wilchek, M. Bioconjugate Chemistry, 4, 1993) as shown in thefirst scheme, FIG. 2B, which demonstrates the attachment of amino acidsto Monomethoxy PEG 2000.

In each of the amino acid attachments, the same peaks in the NMR spectrachanged. The peaks on the proton NMR spectra for the methylene protons(x) of PEG at 4.45 ppm and the succinimidyl ester at 2.56 ppmdisappeared over a period of 60 hours. The same PEG methylene protons(x) appeared at 4.1 ppm in the final amino acid attachment product. Allof the amino acids, arginine, alanine, glycine and 2-fluorophenylglycine, exhibited the same changes in the NMR (data not shown).

The same reactions were carried out with the basic polymer and the sameamino acids, which provided the same results yielding the structuresshown in Scheme 2, of FIG. 2B. The attachment of the EPPT1 peptide tothe polymer using the same chemistry, was then conducted.

An alternative attachment for increasing the percentage of targetingpeptides is to utilize the same chemistry and attach a small percentage(5%) of PEG units to the linker via a selective process to giveadditional reactive PEG hydroxy end groups. The structure of thisselective process is shown in FIG. 2B, third scheme. The PEG units arelocated on the outside of the particle since they are highlyhydrophilic. This would give a much greater number of peptide units perparticle and yet would not disrupt the particle formation.

Example 3 Synthesis of Fluorine-Containing Nanoparticles

Fluorine incorporation into the base copolymer was accomplished usingstandard methods of formation of ester or ether linkages to attach aperfluorinated chain. Each polymer consists of a hydrophilicpolyethylene glycol (PEG) segment (molecular weight main chain 600-1500)bound to a linker (aromatic or peptide bond) to which a hydrophobic sidechain is bound (via ether or ester linkages) that is terminated by ahydrophobic or hydrophilic group. When dissolved in water above thecritical micelle concentration, about 8 to 12 polymeric units selfassemble into a spherical micelle consisting of a compact coresurrounded by an outer envelope of PEG loops that providebiocompatibility. The micelles have a molecular weight of about100-200,000 and a hydraulic radius ranging from about 10 to 30 nm.

The amphiphilic copolymer (with PEG, n=15) was mixed with perfluorooctanoyl chloride under basic conditions to attach the acyl perfluorogroup to the phenolic moiety as the hydrophobic side chain (FIG. 3A).The attachment was confirmed with IR spectroscopy and ¹⁹F-NMR. Thisfluorine-modified polymer formed nanoparticles with Rg about 75 nm asdetermined by static light scattering. It contained 28% (w/w) fluorine,corresponding to about 3,800 ¹⁹F atoms per nanoparticle.

Additional agents can be encapsulated in the core. Micelles, in whichthe side chain is a perfluorocarbon synthesized from perfluoroctylbromide that forms a micelle with 30 nm radius containing 28% (w/v)fluorine has been synthesized. Additional perfluorocarbon cargo can beencapsulated inside each micelle to substantially increase fluorinecontent.

This proof of concept confirmed that fluorine-containing polymers can beproduced, and that they form nanospheres.

Other syntheses attaching fluorine groups are possible. The amphiphiliccopolymers with perfluorocarbon side chains were used to furtherencapsulate 1,1,2,2,-tetrahydro perfluorododecanol (20% w/w) (Schmatizedalso in FIG. 3B) using the same procedure. The amount of perfluorocarboncargo encapsulated by the fluorinated polymer was determined byintegration of fluorine NMR spectra. The entire particle contained 42%(w/w) fluorine, corresponding to almost 6,000 ¹⁹F atoms pernanoparticle. We anticipate that we can increase the loading by at leasta factor of two to 12,000 ¹⁹F atoms per nanoparticle. By assuming a cellvolume of 10³ μm³, we estimate that uptake of 10⁵, 10⁶, 10⁷ or 10⁸ ofthese nanoparticles per cell would lead to cellular fluorineconcentrations of about 2, 20, 200, or 2,000 mM, respectively. The lowerlevel would be adequate for ¹⁹F-MRI in the mouse; the upper levels wouldbe more than enough for imaging humans.

FIG. 3C shows the ¹⁹F Spectra from perfluorocarbon encapsulated by1,1,2,2-tetrahydro perfluorodecanol (referred to herein as basepolymer), as an example of data obtained with one embodiment of anamphiphilic copolymer of this invention. A Bruker/Magnex 600 MHzspectrometer (Martinos Center), with microimaging probe (not spinning)was used. Sample 2a was that of a failed covalent synthesis while sample3 represents encapsulated 20% (w/w) 1,1,2,2-tetrahydro perfluorodecanol,with 19F=15% (w/w) and [19F] in solution=17.3 mg polymer/mL water=140 mM

Example 4 Biological Characterization of Nanospheres Attachment ofFluorescent Probe

Rhodamine B was converted to its acid chloride using oxalyl chloride.Treatment of the polymer (substituted with a decane chain as thehydrophobic group) with this acid chloride and base gave a reaction toform an ester linkage with the CH₂OH groups at the ends of the polymerchains, binding it covalently to the polymer. This attachment did notinterfere with nanosphere formation as shown by light scattering. Cy5.5and the targeting peptide attachment may be accomplished as well, asdescribed hereinbelow.

Size Measurement of Nanoparticles

Multi-angle dynamic light scattering (MADLS) (in addition to 90° DLS)was conducted, together with cryo transmission electron microscopy(cryo-TEM) measurements to characterize the size of the probes. Twotypes of formulations, as shown in Table 1, were conducted. Formulation(1) is the base polymer producing unmodified nanoparticles. Formulation(2) is the fluorine-loaded nanoparticles containing 43% (w/w) fluorine.

TABLE 1 Polymer Components for MADLS and cryo-TEM Side Chain -Cargo:Polymer Formulation Main Chain Linker Side Chain Linker LinkageCargo by weight (1) PEG-600 Isophthalate Decane Ether None NA (2)PEG-600 Isophthalate Perflurocdane Ester 1H,1H,2H,2H- 1:4Perfluorododecanol

Dynamic light scattering data were collected using a BI-200SM Brookhavenlaser light scattering instrument (equipped with a Brookhaven BI 9000 ATdigital correlator) at seven different angles (45°, 60°, 75°, 90°, 105°,120°, and 135°) with multiple runs at each angle per sample (3˜6 runs).Both samples were measured at 25° C. at a concentration of 2.0 mg/mL.The autocorrelation function and the sampling time produced by the BI9000 AT digital correlator were used to fit into an exponential decayfunction so that the characteristic decay time (Γ) was obtained for eachrun at each angle per sample (Brookhaven Website.http://www.bic.com/DLSBasics.html, 2005). Then the characteristic decaytimes at each angle were averaged, and the average decay times wereplotted as a function of the square of the scattering vector q, whereq=[4πn sin(θ/2)]/λ, n=the refractive index of the solution, θ=scatteringangle, λ=the laser wavelength. The slope of the Γ−q2 plot (R2>0.97) gavethe diffusion coefficient (D) of the particles in the solution, and thehydrodynamic particle diameter d was evaluated from Stokes' equationD=kBT/(3πηd), where is kB=Boltzmann constant, T=temperature in Kelvin,and η=liquid viscosity. By this method (Cipelletti, L. and Weitz, D. A.Review of Scientific Instruments, 70: 3214-3221, 1999. Kirsch, S., etal. Journal of Chemical Physics, 104: 1758-1761, 1996), the particlediameter of formulation (1) was 10.2±1.0 nm, of formulation (2) was34.3±1.4 nm.

Cryo-transmission electron microscopy (TEM) images were collected by aJEOL JEM-2200FS Field Emission Electron Microscope. A 2.5 μL aliquot ofsample solution was dropped on a holey carbon film coated copper-carbongrid using a micro pipette. The grid was then blotted with filter paperfor five seconds and immediately dipped into liquid ethane. Then thegrid was transferred into the electron microscope for image collectionusing liquid nitrogen to maintain the temperature. Examples of imagesare shown in FIG. 4 for formulation 1 (A) at a concentration of 44.5mg/mL and formulation 2 (B) at 48.5 mg/mL, respectively. Thenanoparticles accumulate at the interface of the ice and the carbonsubstrate. The fine-grained light gray background is the amorphous icecrystal. The dark gray background is the carbon substrate. FIG. 4B showsvalues obtained for the base polymer alone, and that with encapsulatedfluorocarbon.

Uptake of Fluorescently Labeled Nanoparticles

To demonstrate the ability of the nanoparticles to carry a cargo intocells, INS-1 cells were incubated in vitro with labeled nanospheres,containing Rhodamine B chemically attached to the amphiphilic copolymerat the free ends of the PEG chains, at 37° C. for 35 minutes, washed,and fixed. The cells were then examined with a Zeiss LSM 510 Meta highresolution laser scanning confocal microscope equipped with a 100× oilemersion objective lens. Data was gathered using both the appropriatelaser for imaging Rhodamine B and transmitted light for imaging thecells, and the images were merged into one picture (FIGS. 4D and 4E).Staining may represent localization of the polymer/nanoparticles withinendosomes of the cell. Uptake was quantitated and is presentedgraphically in FIGS. 4C and 4F. INS-1 cells were incubated at 37° C.,with the compound (1 mg/mL), and the uptake was found to betemperature-independent, with a maximum non-selective uptake of 2×10⁸nanoparticles/cell seen.

Drug Delivery with Nanoparticles

In order to determine the capability of the nanospheres to carry drugcargo into a cell, in vivo studies were conducted which demonstrated theefficacy of encapsulated non-steroidal anti-inflammatory drugs such asaspirin and naproxen (Kumar, R., et al. Journal of the American ChemicalSociety, 126: 10640-10644, 2004), in a transdermal application,indicating that the nanospheres were able to carry cargo through theskin.

Acute Systemic Toxicity

Acute oral toxicity testing of intact nanospheres and individualcomponents was carried out to determine the LD50 (median lethal dose)with C57Bl/6 (Table 2). For all but one case, the LD50 was far above 2g/Kg, the limit for essentially non-toxic substances (Botham, P. A.Toxicology in Vitro, 18: 227-230, 2004; NIH Guidance Document on UsingIn Vitro Data to Estimate In Vivo Starting Doses for Acute SystemicToxicity, NIH Publ 01-4500. pp. 48. Research Triangle Park, N.C., USA:NIEHS, 2001). Even the most toxic nanospheres in this study,(isophthalate linker, ether linkage), gave an LD50 of 1 g/Kg, which ishigher than that for many food additives on the list of those generallyregarded as safe (GRAS) (NIH Guidance Document, supra; EAFUS: A FoodAdditive Database. FDA/Center for Food Safety & Applied Nutrition,2004).

TABLE 2 Acute oral toxicity tests Hydrophilic Linkage Group Linker BondSide Chain LD50 (g/Kg) PEG 600 38 Aspartic Acid 20 PEG 600 Aspartic AcidAmide Nonyl 60 PEG 600 Isophthalate Ester Decane 60 PEG 600 IsophthalateEther Nonyl 1

Cellular cytotoxicity following exposure to the polymers and reagents:INS-1 cells were incubated with the polymers and/or reagents (1 mg/mLpolymer or 0.05 mg/mL dye) at 37° C., and cellular viability wasdetermined by MTS (FIG. 5). Base polymer or encapsulated rhodamineexposure did not induce any observable cytotoxicity, up to 48 hourspost-exposure, though cytotoxic effects were readily observed even 3hours following exposure to Rhodamine alone.

INS cells incubated with 0.2 mg/mL free or encapsulated doxorubicin at37° C., showed a comparable rate of cell death, with the death ratedecreasing with the presence of PFC side chains, as measured by MTS.Encapsulation of doxorubicin resulted in reduced cytotoxicity in U87cells, with BP, but not PFC side chains.

The kinetics and sensitivity of detection of cellular uptake wereevaluated as well (FIG. 6). INS-1 cells were incubated with 1 mg/mLpolymer at 37° C. and evaluated by the indicated reader. The sensitivityof the plate reader was low as the background created too much noise.Data obtained using Cellomics ArrayScan was less sensitive to backgroundnoise and allowed for quantification of cellular uptake, which increasedrapidly, then levelled out after 4 hours (FIG. 6A). Confocal microscopicevaluation of INS-1 cells incubated with 0.2 mg/mL BP-RB for 30 min or 1mg/mL BP-PFC-FITC for 14 hr, washed 3× (2 min), and fixed (20 min) (FIG.6B), showed that the fluorescent polymer was essentially confined tocytoplasmic vesicles and not the nucleus.

Uptake of free and encapsulated Doxorubicin was evaluated by confocalmicroscopy as well (FIG. 6C). Cells were incubated with 0.2 mg/mL freeor encapsulated doxorubicin for 5 hr at 37° C., washed 3× (2 min), andfixed (20 min). Doxorubicin was largely confined to the nucleus.Therefore, doxorubicin must be released from the polymer, since thepolymer is too large to enter the nucleus.

Example 5 Targeting of uMUC-1 Antigen with a Multi-Modal Imaging Probe

Nanoparticles are more easily taken up by a tumor as compared to normaltissue, because of the generally greater vascularization andinterstitial volume of a tumor. However, in order to determine whetherit is possible to greatly enhance selectivity, a ligand may be coupledto the free ends (16-24 per micelle) of the PEG-linker segments and, inanother embodiment, to functional groups introduced into the PEG chains.

The ligand initially chosen was a 15-amino acid synthetic peptidedesignated EPPT1 that is derived from the binding site of a monoclonalantibody raised against human epithelial cancer cells displaying theunderglycosylated mucin-1 antigen (uMUC-1). MUC-1 is a transmembranemolecule expressed over the cell surface and in internal compartments bymost glandular epithelial cells. It is overexpressed on almost all humanepithelial cell adenocarcinomas as well as some nonepithelial andhematological malignancies (altogether accounting for more than 70% ofall newly diagnosed cancer cases) in an underglycosylated form, whichexposes an immunogenic epitope that is normally masked. The syntheticpeptide EPPT1 has a reasonably high binding constant (K_(d)=20 μM), andnanoparticles bound to the epitope would either remain bound to the cellsurface or be internalized by receptor-mediated endocytosis. In thisway, selectivity is determined primarily by the specific ligand-receptorinteraction rather than by the ease of perfusion through the tumor.

A multi-modal imaging probe targeting uMUC-1 tumor antigen wassynthesized and tested both in vitro and in vivo (Moore, A., et al.Cancer Research, 64: 1821-1827, 2004). The probe consisted ofcross-linked iron oxide as an MR-imaging contrast agent that carriedCy5.5 dye as a NIRF optical probe and EPPT1 peptides produced bysolid-phase synthesis, both attached to amino groups linked to the CLIOdextran coat. A FITC label was added to the NH, terminus of the peptidefor subsequent fluorescence microscopy analysis, and the probe wasradioiodinated by attachment to the peptide tyrosines by the Iodogenmethod for cell binding analysis and biodistribution studies (FIG. 7).

A terpolymer (FIG. 7, Series 1) was obtained via enzymaticpolymerization using novozyme-435, 5-amino dimethylphthalate, 5-hydroxydimethylphthalate and polyethylene glycol. FITC and Cy5.5 were attachedto the polymer by stirring separately in dimethylformamide for fourhours at room temperature with the terpolymer. The resultant polymer wasdialysed and used for'partial o-alkylation using alpha bromo acetyltriethyleneglycol in K₂CO₃ and acetonitrile on refluxing them together.The free hydroxyl group at the end of the triethyleneglycol unit of thepartially alkylated polymer was activated by stirring disuccinimidylcarbonate in acetonitrile with DMAP and used for peptide attachment. Theactivated hydroxyl polymer was stirred with peptide in phosphate buffer(pH-7.2) for 12 hours to obtain the desired polymer. A hydrocarbon chainwas then introduced by stirring the halogenated hydrocarbon chain withpolymer in triethyl amine to make the carrier molecule suitable formicelle formation.

To attach FITC to the Terpolymer, FITC was added to a three necked roundbottom flask containing terpolymer dissolved in anhydrous DMF under theenvironment of nitrogen. The resulting mixture was stirred at roomtemperature for four hours, after which DMF was washed out using anexcess of hexane. The remaining residue was dried under vacuum. Residuewas then subjected to dialysis (6000-8000 Mw dialysis bag) to removeunreacted FITC in the product. The resultant product was characterizedfrom its ¹HNMR and UV spectra.

To attach Cy5.5 to the Terpolymer, Cy5.5 was added to a three neckedround bottom flask containing terpolymer dissolved in anhydrous DMFunder the environment of nitrogen. The resulting mixture was stirred atroom temperature for four hours. After the completion of the reactionDMF was removed by washing several times with an excess of hexane. Theresidue was further dried under vacuum. Residue was then subjected todialysis (6000-8000 Mw) to get rid of unreacted Cy5.5 in the obtainedproduct. The resultant product was characterized from its ¹HNMR and UVspectra.

For the esterification of free hydroxyl on the terpolymer, nonanoylchloride dissolved in dichloromethane was added dropwise in reactionmixture containing dye-attached terpolymer and triethylamine, undernitrogen with constant stirring at room temperature. The resultingmixture was stirred for six hours. After completion of the reaction,solvent was removed under vacuum and THF was added and then filtered toremove salt formed in the reaction. Unreacted nananoyl chloride wasremoved by washing with hexane. Obtained product was dried under vacuumand was characterized on the basis of its ¹HNMR and UV spectrum. Asimilar method was used for polymers with FITC and polymers with Cy5.5.

For O-alkylation, the polymer was dissolved in anhydrous acetonitrileand added to three neck round bottom flask under nitrogen with constantstirring containing fused K₂CO₃, followed by dropwise addition ofbromoester of TEG dissolved in acetonitrile. The resulting mixture wasrefluxed for eight hours. After completion of the reaction K₂CO₃ wasfiltered off and filtrate obtained was concentrated under vacuum to getthe desired product.

For activation of the free hydroxyl group, disuccinimidyl carbonate andthe polymer dissolved in acetonitrile in the presence of DMAP werestirred in a nitrogen environment, and the resulting mixture was stirredfor 12 hours. Solvent was then removed under vacuum at room temperature.Separated salts and N-hydroxyl succinamides were removed by repeatedprecipitation with diethyl ether in acetonitrile. The thus obtained purecompound was vacuum dried, characterized, and used for peptideattachment.

For peptide attachment, an activated hydroxyl polymer was stirred withpeptide in phosphate buffer (pH-7.2) for 12 hours to get the desiredpeptide attached polymer. The product was characterized by NMR, IR andUV spectroscopy. After the peptide was attached, the hydrocarbon chainwas attached in order to make the polymer suitable for micelleformation. This was achieved by stirring the halogenated hydrocarbonchain with polymer in triethylamine.

To extend these studies using perfluoro labeled polymer, the sameFITC-labeled EPPT1 peptide was used. The crosslinked iron oxide(CLIO)-EPPT probes had a hydrodynamic diameter measured by dynamic lightscattering of about 36 nm, slightly smaller than fluorine-containingmicelle nanoparticles, and contained 14 peptides and 5 Cy5.5 moleculesper particle, comparable to what is anticipated with the micelles. Thestudy with the CLIO-EPPT nanoparticles established test procedures whichwill be used.

EPPT1 peptides were attached to the free PEG terminal hydroxyls usingcarbodiimide chemistry: a stoichiometric excess of N-hydroxysuccinimideand carbodiimide are added to the micelles in water, reacted for 15-30minutes at room temperature, and purified by dialysis. The EPPT1peptides were added to the modified polymer and reacted overnight atroom temperature. The reaction occurs between —OH on the PEG unit andthe N terminus (—NH₂) on the peptide. Purification of the probe isaccomplished by dialysis or column separation.

Cell binding assays (FIG. 7D) were carried out with a variety of humanuMUC-1-positive tumor cell lines: ZR-75-1 (breast), BT-20 (breast), HT29(colon), CAPAN-2 (pancreas), LS174T (colon), and ChaGo-K-1 (lung) aswell as human control uMUC-1-negative tumor and normal cell lines. Celllines were incubated with varying amount of ¹²⁵I-labeled CLIO-EPPT for 1hour. uMUC-1-negative and normal cell lines had much lower nanoparticleuptake than that of the uMUC-1-positive tumor cell lines, which bound onthe order of 10⁷-10⁸ CLIO-EPPT nanoparticles per cell.

The same lines and protocols will be used for determiningfluorine-loaded micelle nanoparticle uptake, with 10-1000 or higher mMfluorine concentration uptake in the uMUC-1-positive cells being theconcentration sought. Similar binding assays will be conducted, andbinding as a function of both concentration and time will be determined,in order to explore conditions that provide maximum selectivity betweenuMUC-1-positive tumors and normal cells.

In vitro specificity of CLIO-EPPT for adenocarcinomas was furthercharacterized by flow cytometric analysis of probe binding to selectedadenocarcinoma and control cell lines (FIG. 7E). A control cell lineshowed no cell binding, whereas adenocarcinoma cell lines bound theprobe and displayed diverse staining intensities, consistent withvariable glycosylation. Fluorescence microscopy experiments, in whichadenocarcinoma and control cells were incubated with the probe,confirmed the fluorescence-activated cell sorting data. All of theadenocarcinoma cell lines stained strongly and showed colocalization ofthe FITC and Cy5.5 signals.

In vivo ¹H-MR imaging was performed on animals bearing bilateraluMUC-1-positive and uMUC-1-negative tumors before and 24 hours afterprobe injection. No significant change in signal intensity ofT2-weighted images was observed in uMUC-1-negative tumors, whereassignificant signal reduction was observed in some regions ofuMUC-1-positive tumors (a 52% decrease for LS174T tumors versus a 13-18%decrease in control tumors, FIG. 8A). The same animals were subjected tooptical imaging immediately after the MR-imaging session. A highintensity NIRF signal was obtained from the uMUC-1-positive tumors,whereas no significant signal was observed from the control tumors(FIGS. 8B and 8C). Fluorescence microscopy of excised tumors and muscletissue gave results consistent with NIRF images. From biodistributionstudies with ¹²⁵I-CLIO-EPPT, on average uMUC-1-positive tumorsaccumulated 3.4 times more of the probe than uMUC-1-negative tumors.Correlative dual channel fluorescence microscopy of excised tumorsshowed colocalization of FITC and Cy5.5 signals in uMUC-1-positivetumors but no signal in control tumors (FIG. 8D).

A multi-modal probe for MR and NIRF imaging was herein characterized andtested in vitro and in vivo in animal models of human cancer, indicatingspecific accumulation in uMUC-1-expressing tumors and providing in vivoimaging results.

Similarly, nanoparticles carrying perfluorocarbons will be used for¹⁹F-MR imaging, which has some advantages over ¹H-MR imaging.Cell-associated fluorine concentrations necessary to make use of theseadvantages are obtainable, and versatile imaging probes can be developedaccording to the methods and processes of this invention, that may beused for the detection of cancer, inter-alia, and, in other embodiments,monitoring the progression of intervention in afflicted subjects.

Example 6 Synthesis and Characterization of Multi-Modal Probes for InVivo Cancer Imaging

Probes comprising a perfluorinated polymer with Cy5.5 attached for NIRstudy as well as attachment of the targeting peptide EPPT1 to thehydroxyl groups at the free ends of the PEG chains may be synthesized asdescribed herein.

Synthesis of the polymer backbone will be carried out using awell-established enzymatic method (Kumar, R., et al., Journal ofMacromolecular Science, 2002, supra; Kumar, R., et al. Journal of theAmerican Chemical Society, 2004, supra). The synthetic scheme is shownin FIG. 9. Polymerization involves two linkers with linker 2 present insmall amounts (1-5%) to produce the polymer shown. The synthesis isconducted such that PEG hydroxy groups are present at the ends of thechain.

Perfluorocarbon side chains are then attached to the linker hydroxylsusing standard acylation procedures to form ester linkages with thepolymer backbone. Other linkers, such as, for example, ether amide, maysimilarly be utilized.

A variety of fluorine-containing polymers may be prepared via thisscheme, as exemplified in FIG. 10. The synthetic schemes may beconducted using amine-modified base polymer, while the inclusion ofsmall amounts of the amine moiety should not interfere with nanosphereformation.

In addition, chain length may be varied (from 5 to 13 carbons), and therelative number of fluorine atoms may be varied, which may alter thehydrophobicity of the side chain.

The synthesis and in vitro characterization studies describedhereinabove may be used for optimization of nanoparticle formulation.The effect of synthesis parameters on loading and stability may also bestudied, and a number of encapsulating materials may be investigated,including perfluorodecalin, bromo-perfluoroheptane, and perfluoro-crownether. Cy5.5 may be attached, in order to perform NIRF determination,EPPT1 peptides may be used for targeting, and radioiodine may beincorporated for cell binding and biodistribution studies. A number offormulations may be tested to determine specificity and cellular uptake.

Synthesis of the polymer labeled with Cy5.5 dye may be conducted asdescribed (Josephson, L., et al. Bioconjugate Chemistry, 10: 186-191,1999), using aminated CLIO nanoparticles, or partially aminated basicpolymer. The attachment of the Cy5.5 monofunctional dye to amine groupsof the base polymer is performed by adding 100 mg of polymer in 0.5 Msodium bicarbonate with the pH adjusted to 9.6 to 1 mg of Cy5.5 dye(Amersham-Pharmacia, Cat. #Q15408). The mixture is incubated on arotator overnight at room temperature. After incubation, the mixture ispurified from non-reacted dye on a G-25 Sephadex column equilibratedwith 20 mM sodium citrate buffer with 0.15 M NaCl, pH 8.0. Incubationtimes are varied to achieve different polymer:Cy5.5 ratios.

The EPPT1 peptide, YCAREPPTRTFAYWG (SEQ ID NO: 1) may be modified tointroduce a FITC label for subsequent fluorescence microscopy/FACSanalysis. The FITC label will be introduced by adding FITC-labeled Lyson the C-terminus followed by a second unlabeled lysine to serve as anattachment point. The Cys thiol group will be protected with an acetoxymethyl group. The final peptide will have the following sequence:Y-C(ACM)-A-R-E-P-P-T-R-T-F-A-Y-W-G-K(FITC)K (SEQ ID NO: 2) (Italic fontindicates the original sequence). The peptide is synthesized on anautomatic synthesizer (PS3, Rainin, Woburn, Mass.) using Fmoc chemistrywith HBTU and HOBT. Peptides are cleaved from the Rink amide HBHA resin(Novabiochem, San Diego, Calif.) with 5 ml ofTFA/thioanisole/ethanedithiol/anisole (90/5/3/2) and purified by C18reverse phase HPLC. Molecular weight is determined by MALDI massspectroscopy.

The synthesis of Cy5.5-EPPT1 nanoparticles is accomplished via theattachment of the FITC-labeled peptides to the Cy5.5-modified basicpolymer, via the procedure of Zalipsky, et. al. (Zalipsky, S., et al.Biotechnology and Applied Biochemistry, 15: 100-114, 1992), whichattaches proteins through succinimidyl activation of the PEG hydroxygroup to react with the N-terminus of the peptide, in this case, theadded lysine group.

A number of other methods for attaching the peptide are also envisioned(see, for example, Roberts, et. al. (Roberts, M. J., et al. AdvancedDrug Delivery Reviews, 54: 459-476, 2002).

In order to determine cell binding and biodistribution, the probe may beradiolabeled with Na ¹²⁵I with the Iodogen method (Pierce, Rockford,Ill.) using available Tyr within the peptide sequence. The basic polymerbackbone itself may be radiolabeled with the same procedure because theisophthalate ring is substitutable in the same manner as the tyrosinearomatic ring. Nanospheres may have a substituted phenolic aromatic ringas the linker in polymeric backbone which has the positions ortho andpara to the substituted hydroxy group available for substitution, asshown in FIG. 11A. The positions indicated by the arrows would beavailable to substitute radiolabeled iodine. Utilization of commerciallyavailable kits would give single or multiple substitutions on thesearomatic rings. The EPPT1 peptide also has a tyrosine moiety which wouldalso be susceptible to iodine substitution via the same procedures. Thenumber of iodines bound per polymer chain (i.e. specific activity) iscontrolled by the radioiodine concentration and reaction time.Substitution at many of these active sites would provide a relatively“hot” sample which could be utilized for in vivo imaging or RIT studiesin mice, if desired.

It is also possible to encapsulate chelated yttrium and indium byappropriately modifying the nanospheres. These radionuclides may beutilized with the nanospheres containing the targeting peptides tofurther enhance the sensitivity of the imaging with radioactive nuclei.Technetium may also be similarly used.

In order to track the encapsulated cargo of the nanoparticles,fluorescent labeling of perfluorocarbon is desirable. In one embodiment,Rhodamine B is converted to its acid chloride form and treated with1,1,2,2 tetrahydro perfluorododecanol to form the esterified perfluorocompound. This will then be encapsulated with the perfluorinated basicpolymer. This modification will allow tracking of the cargo byfluorescence microscopy.

It is also possible to synthesize polymers with labeled perfluorinatedside chains as shown in FIG. 11B. The synthesis of Rhodamine B-labeledperfluorinated bromo compounds utilizes enzymatic synthesis involvingthe lipase Novozyme-435. It is possible to selectively monoacylate anumber of compounds with bromoacetic acid. Alkylation of the phenolichydroxyl group of the polymer with the Rhodamine B-labeledperfluororinated bromo compound results in the desired polymer withlabeled perfluorinated side chains. This sequence represents anotherembodiment for a method of attaching the fluorinated groups to thepolymer backbone.

The final polymer structure(s) is, in one embodiment, the sum of theabove syntheses as shown in FIG. 11.

The final structures may then be analyzed for (1) elemental analysis(Galbraith, Knoxville, Tenn.) of loaded and not-loaded nanoparticles todetermine fluorine content, (2) number of Cy5.5 molecules, (3) number ofpeptides, (4) particle size (N4-MD, Coulter), and (5) isotope bindingyield. Absorption spectra of the probe will be taken using a Hitachi3500 spectrophotometer (Amax for Cy5.5 is at 675 nm). The synthesesdescribed may be adjusted in order to optimize these parameters.

The morphology of the micelles and the size of the corona and core maybe visualized by cryogenic transmission electron microscopy according tothe method described (Lam, Y. M., et al. Molecular Simulation, 30:239-247, 2004). In order to form a monolayer of micelles, which isoptimal for observation under a transmission electron microscope,aqueous solutions of varying concentrations is vitrified. Approximately2 μL of solution is applied to a carbon grid while the system ismaintained at 40° C. in a high humidity freezing apparatus for 30 s. Thegrid is blotted with a double layer of filter paper for 5-10 seconds andthen plunged into liquid ethane. The vitrified sample is placed in acryotransfer holder maintained at liquid nitrogen temperature ˜170° C.TEM images of the sample will then be recorded on a high resolutioncryo-electron microscope (JEOL 2200 FS) available at the Whitehead-MITBioimaging Center. The size and shape of the micelles are observed andqualitative and quantitative assessments made.

Example 7 Specificity and Cellular Uptake of the Candidate Probes byuMUC-1-Positive and uMUC-1-Negative Tumor Cell Lines

For targeted multi-modal imaging probes labeling of tumor cells to be“visible” for the imaging systems using ¹⁹F-MR imaging, it is necessaryto attain a sufficiently high cell-associated probe concentration, alarge number of binding sites for the EPPT1 peptide, a rapid rate ofaccumulation, high specificity, etc.

In order to measure the amount of cell-associated nanoparticle probes(and by inference the amount of fluorine) as a function of the probesolution concentration and incubation time, cell lines having differentdisseminating potential may be used, since the probe should accumulatenot only in primary tumors but also in metastatic sites. Probespecificity may be determined by comparing probe accumulation resultsfor normal cells, uMUC-1-positive and uMUC-1-negative tumor cells. Cellviability assays may be performed with cells having maximal cellularaccumulation of probes to investigate effects on cell viability.

The probe is radiolabelled with Na ¹²⁵I using the Iodogen method(Pierce, Rockford, Ill.) using available Tyr within the EPPT1 peptide.Cell lines used may be, for example, those listed in Table 3.

TABLE 3 Cell lines used for measurement of cellular accumulation ofprobes Cell line Tissue MUC-1 expression CAPAN-2 pancreas + LS174Tcolon + ChaGo-K-1 lung + NCI-H661 lung; metastatic site: lymph node −ZR-75-1 breast + BT-20 breast + MCF10-A normal breast; fibrocysticdisease − RF-1 stomach + RF-48 stomach; metastatic site: ascites −OVCAR-3 ovary + DU-145 prostate + LNCaP prostate − U87 glioma − 293primary embryonic kidney − primary; peritoneum − macrophageCells are incubated with increasing concentrations of ¹²⁵I-labeled probefor different time periods at 37° C. in a humidified CO₂ atmosphere,followed by extensive washing with HBSS. After the final wash, cells arelysed with 0.1% Triton X, and cell lysates are counted in a gammacounter (1289 Compugamma LS; Wallac, Turku, Finland). The relationshipbetween ¹²⁵I radioactivity and probe are measured using solutions withknown probe concentrations. Cell number or cell concentration areestimated by flow cytometry to calculate the amount of probes associatedper cell after each measurement

Once the amount of fluorine accumulation in 10 million cells is withinthe detection limit of MRI, then ¹⁹F-MRI phantoms are prepared usingcell pellets. Selected cells may be subjected to cell viability assayswith MIT (mitochondrial function), caspase activation (apoptosis), and7-AAD (membrane integrity).

Differential binding of targeted nanoparticles to adenocarcinoma(CAPAN-2, HT-29, LS174T, BT-20, and ChaGo-K-1) versus control (MCF10A,293, and U87) cell lines may also evaluated using fluorescencemicroscopy. Cells are grown overnight on coverslips, fixed in 4%paraformaldehyde, incubated with targeted probes, and washed. Cells areidentified under a bright-field microscope and then subjected tocorrelative dual-channel fluorescence microscopy in the green (for FITCdetection) and NIR (for Cy5.5 detection) channels, using an invertedfluorescent microscope (Zeiss Axiovert 100TV, Zeiss, Wetzlar, Germany).Images are collected using a cooled charge-coupled device (Photometrics,Tucson, Ariz.) with appropriate excitation and emission filters (OmegaOptical, Brattleboro, Vt.).

The Cy5.5 and FITC fluorescence intensity in cell lysates and in knownsamples containing known probe concentrations are measured, for example,with a plate reader (BMG PolarStar, BMG Labtech, Offenburg, Germany).Scatchard plot analysis is used to estimate R_(T), the number ofreceptors per cell, and K_(D,eff), the effective affinity of the probefor the cell. Optimization of these procedures such that probeaccumulation is sufficient for MR and/or NIRF imaging, with theScatchard plot analysis providing an estimate of the effective affinityof the multivalent probe for the cell (Schwartz, A. L., et al. Journalof Biological Chemistry, 257: 4230-4237, 1982).

Example 8 Temporal Cellular Distribution of the Candidate Probes

In order to optimize nanoparticle uptake, retaining their highspecificity, fluorescently labeled perfluorocarbon-containingnanoparticles are evaluated for nanoparticle distribution andnanoparticle disintegration in uMUC-1-positive and uMUC-1-negative tumorcell lines and normal cells.

Cells are seeded in 35 mm cell culture dishes and six-well platescontaining 22 mm-diameter glass cover slips at a density of 1.5×10⁵cell/mL and incubated with nanoparticle concentrations that give maximaluptake, as described in Example 7. Laser scanning confocal micrographswill be recorded using, for example, a Zeiss LSM 510 Metahigh-resolution laser scanning confocal microscope (Carl Zeiss AG).Scanning speed and laser intensity will be adjusted to avoidphotobleaching of the fluorescent probes and damage or morphologicalchanges of the cells. The microscope will be equipped with amicrocultivation system (Incubator S, CTI controller 3700 digital,Zeiss) to control temperature, humidity and CO² for maintainingphysiological conditions during experiments. Image analysis andfluorescence signal quantification will be performed using, for example,Zeiss LSM software.

In order to investigate subcellular probe distribution, selectivelabeling of different cellular organelles will be conducted as described(Savic, R., et al. Science, 300: 615-618, 2003): (1) plasma membranewith 5-dodecanoylaminofluorescein (DAF, green), (2) nuclei with Hoechst33342 (blue), (3) lysosomes with lysotracker DND-26 (green), (4)mitochondria with Mito-Tracker Green FM (green), (5) Golgi apparatus andendoplasmic reticulum (ER) with Brefeldin A BODIPY FL conjugate (green),and (6) mitochondria and ER with 3,3′-dihexyloxacarbocyanine iodide(green). When using green dyes for labeling organelles, it will benecessary to remove FITC from the EPPT1 peptide. Probes with no peptideswill serve as controls for all experiments.

A spinning-disk confocal microscope, such as, for example, the PerkinElmer PE Ultraview RS100, equipped with a high speed digital cooled CCDcamera with a 1.5 s scan time for 3-D images that is suitable forproducing time-lapse video of rapid events may similarly be used.

Example 9 Biological Characterization of Multi-Modal Imaging Probes

In order to determine the accumulation of the probe in different organs,and/or the signal to noise ratio for MR imaging of the tumors abi-lateral tumor propagation method (Weissleder, R., et al. NatureMedicine, 6: 351-354, 2000; Moore, A., et al. Radiology, 221: 751-758,2001) will be used, where uMUC-1-positive tumor will be injected in oneflank of the mouse and uMUC-1-negative tumor will be injected in theopposite flank, and include tumors with different metastatic potential.The accumulation of the probe at primary and metastatic sites isevaluated.

Animals are anesthetized with an intraperitoneal injection ofketamine/xylazine (80 mg/kg/12 mg/kg, Parke-Davis. Morris Plains,N.J./Miles Inc., Shawnee Mission, Kans.). Tumor cells (uMUC-1+ anduMUC-1−) are injected in the flanks of nu/nu mice (n=5/pair;approximately 5×10⁶ cells/flank depending on the tumor doubling time).Tumors are allowed to grow to 0.5 cm in size, and animals are injectedintravenously with ¹²⁵I-labeled ¹⁹F nanoparticle probes. Animals aresacrificed, for example, 24, 48 and 72 hours later by lethal IVinjection of sodium pentobarbital (200 mg/kg). Tumors, tumor metastasis,the pancreas, spleen, liver, heart, intestine, lung, lymph nodes,thymus, blood, bone, muscle, brain and fat are excised, weighed andradioactivity is measured in a gamma counter. Aliquots of the probes arecounted simultaneously to correct for radioactive decay and to calculatethe dose in each organ. Biodistribution results are expressed as thepercentage of the injected dose per gram of tissue (% ID/g).

¹²⁵I-labeled probe in tumor bearing animals (n=5) is assessed. Bloodhalf-life of the probe is determined after intravenous injection of 50mCi/animal of the ¹²⁵I-labeled ¹⁹F nanoparticles. Blood samples arewithdrawn over several time points from the tail vein, weighed, andradioactivity in the blood is counted in a gamma-counter. Bloodhalf-life is calculated as described (Ritschel, W. Handbook on BasicPharmacokinetics, 3d edition, p. 168-190. Hamilton, Ill.: DrugIntelligence Publications, Inc., 1986). The preparation of Cy5.5-labeled19F nanoparticles with no peptides attached and/or with nonsense peptideserve as controls for all studies.

Results with acute oral toxicity testing indicated that the nanosphereswith a hydrocarbon side chain have little or no toxicity. Thenanoparticles for ¹⁹F-MR imaging contain perfluorocarbons, which havebeen used extensively in artificial blood applications, and most aregenerally viewed as biologically inert. An acute lethality (LD50) testmay be used to determine if the greater concentrations used in theapplications of this invention result in toxicity.

A basal cytotoxicity test may be initially conducted to predict astarting dose for an in vivo lethality test. The neutral red uptake(NRU) test may be undertaken with BALB/c 3T3 cells (NIH GuidanceDocument on Using In Vitro Data to Estimate In Vivo Starting Doses forAcute Systemic Toxicity, NIH Publ 01-4500. pp. 48. Research TrianglePark, N.C., USA: NIEHS, 2001). NR is a weak cationic dye that readilypenetrates cell membranes by non-ionic diffusion and accumulatesintracellularly in lysosomes. Alterations of the cell surface or thesensitive lysosomal membrane lead to lysosomal fragility and otherchanges that gradually become irreversible, leading to a decreaseduptake and binding of NR. It is thus possible to distinguish betweenviable, damaged, or dead cells, which is the basis of this assay.Healthy BALB/c 3T3 cells, when maintained in culture, continuouslydivide and multiply over time. A toxic chemical, regardless of site ormechanism of action, will interfere with this process and result in areduction of the growth rate as reflected by cell number. Cytotoxicityis expressed as a concentration dependent reduction of the uptake of thevital dye, NR, after one day (one cell cycle) of chemical exposure, thusproviding a sensitive, integrated signal of both cell integrity andgrowth inhibition.

BALB/c 3T3 cells are seeded into 96-well plates and maintained inculture for 24 hours to form a semi-confluent monolayer. Cells areexposed to the nanoparticles over a range of concentrations. After 24hours exposure, NRU is determined for each treatment concentration andcompared to that of control cultures. For each concentration of the testchemical, the percent inhibition of growth is calculated. The IC₅₀ (theconcentration producing 50% reduction of NR uptake) is calculated fromthe concentration-response and used to estimate the starting dose forlethality test.

In one embodiment, an acute lethality test may comprise targetednanoparticle administration into the tail vein of 20-25 gram ICR mice.Different dose levels are used, and a number of animals are given eachdose. The animals are observed for, for example, 14 days, with the LD₅₀determined by the Reed-Muench method (Reed, L. J. and Muench, H. Am JHyg, 27: 493-494, 1938), and the safety factor calculated as the ratioof LD₅₀ to the effective dose.

Example 10 In Vivo Imaging of uMUC-1-Expressing Human Tumors

¹⁹F MR spectral characteristics of nanoparticles taken up in cells suchthat optimal imaging is achieved, may nonetheless be influenced bychanges in the ¹⁹F MR spectrum, which negatively affect image formation(for example, resonance line-widths must not be substantially broadenedon binding such that the T2* becomes too short for imaging).

Toward this end, uMUC-1-positive tumors are grown in animals asdescribed hereinabove, after which animals are injected intravenouslywith non-radiolabeled ¹⁹F nanoparticle probes and sacrificed. Tumors andmetastases are harvested and analyzed ex vivo by ¹⁹F NMR spectroscopy at14 T field strength, 37° C. For each specimen, single pulse static(non-spinning) and 2.5 kHz magic angle spinning (MAS) spectra areobtained. T1 is measured by inversion recovery and T2 measured by CPMGin MAS spectra. The static spectra yields the best estimate of theappearance of the in vivo spectrum, and is used to calculate T2*(=1/π∘FWHH) for each resolved chemical shift band.

By tabulating T2, T2* and T1, an early predictor for the performance ofthe nanoparticles under in vivo imaging conditions is obtained. The T2*(a measure of the inverse line-width) of any resonance must not be belowthe order of a few ms in order for imaging to be successful based onthat resonance. The T2* will be affected by the degree of molecularmotion, with nanoparticle binding potentially creating T2* values whichare too short for particular resonances to be used for image creation.Shortening of T1 on binding or aggregation, improves image signal tonoise ratio, making it useful to identify resonances with advantageouslyshort T1s. Although chemical shift selective pulses have been used in¹⁹F MRI in order to select out of a complicated chemical shift spectrumjust a single resonance that is used for image creation, this results inmuch (or most) of the potentially available fluorine signal to bediscarded.

The determination of T2, T2* and T1 for each resonance band enables anassessment of which resonances in the chemical shift spectrum can beprofitably used in image creation.

The magic angle spinning spectra of the tissue specimens yield thehighest spectral resolution and lowest detection limits because thespinning eliminates isotropic magnetic susceptibility broadening effects(Cheng, L. L., et al. Magnetic Resonance in Medicine, 36: 653-658,1996). MAS spectroscopy has become the standard for measurement ofproton NMR spectra of tissue specimens. Each resonance in the PFC chainis resolvable with this technique, and hence amenable to assessment forits contribution to image creation. Interestingly, despite theavailability of this technique in tissue NMR spectroscopy for almost adecade, there have been no reports of its use for ¹⁹F spectroscopy oftissue specimens. Although ¹⁹F generally has much larger chemical shiftsthan protons, making the elimination of susceptibility broadeningpotentially less important for most applications of ¹⁹F tissuespectroscopy, the severe crowding and complexity of the CF2 resonancesin the nanoparticle spectra may accrue substantial benefits from MASspectroscopy.

In order to determine whether the nanoparticle molecular probes of theinvention can successfully label tumors for detection by ¹⁹F-MRI, invivo imaging is conducted. uMUC-1-positive tumors grown in animals to0.5 cm in size, are injected intravenously with nonradiolabeled ¹⁹Fnanoparticle probes and scanned by ¹H and ¹⁹F-MRI, at, for example, 24,48 and 72 hours post injection. Scanning is performed with, for example,a multinuclear Bruker (Karlsruhe, Germany and Billerica, Mass.) AvanceNMR console interfaced to a 14 T (600 MHz proton frequency) Magnex(Oxford, UK) actively shielded 89 mm vertical superconducting magnet.Animals are physiologically supported (if necessitated by the verticalpositioning and long exam time) with a ventilation system and monitoredfor respiration, core temperature, and ECG. The animals are suspendedhead-up (by a bite bar arrangement) in a Bruker Micro 2.5 microimagingprobe. A Teflon-free dedicated Bruker 19F 30 mm cylindrical RF resonatoris used for excitation and detection of both ¹H and ¹⁹F images. Thisresonator, although optimized for ¹⁹F MRI, will also tune to ¹H withoutremoval of the animal from the magnet and performs proton MRI.

The anesthetized animal is positioned in the magnet, stabilized, with atriplane proton scout image used to roughly locate the tumor area.Multi-slice proton T1-weighted spin echo and T2-weighted gradient echoimages (256×256 matrix size, 0.5 mm slice thickness) are obtained todelineate the tumor and surrounding area. The resonator is tuned to the¹⁹F frequency (564 MHz) and imaged by ¹⁹F MRI as described below. The¹⁹F images (showing the nanoparticle distribution) are overlaid on the¹H images (which delineate detailed anatomy).

Because of the lower signal to noise ratio of the ¹⁹F signal and thebroad chemical shift range, all ¹⁹F images are non-slice selective(projective). Several MRI techniques are explored. In order to captureas much of the available ¹⁹F signal as possible, chemical shiftselective pulses may not be used. The most direct method for total-¹⁹FMRI is to use small magnetic field gradients, which do not cause overlapof projections from different chemical shift bands. In the case of thenanoparticle probe spectra obtained at 14 T under in vivo-likeconditions (using the imaging probe and a low resolution sample holder),the linewidths of the CF3 and CF2 bands are about 0.5 ppm (based onspectra we obtained with the nanoparticles). This linewidth arisesprimarily from unresolved J-couplings in the CF3 band and the bands fromCF2 adjacent to either CF3 or the polymer linkage (at 85, 40 and 49 ppmfrom C6F6 respectively). The remaining CF2 band centered at 44 ppmcontains multiple chemical shifts as well as the couplings. A 5 ppmprojection width will just barely avoid overlap of these bands, and willprovide 10 pixels of linear image resolution across the subject, aspatial resolution comparable to the true resolution obtained in otherin vivo ¹⁹F applications in the literature. Use of a larger gradientresults in higher spatial resolution, but lower signal to noise ratio.This method can be applied to both projection (radial) reconstructionand phase encoded approaches toward building a 2D image.

A second approach may use mathematical deconvolution to remove theresolution-degrading effects of the chemical shift spectrum from theimage. It requires that the chemical shift spectrum be constant in shape(although it may vary in intensity) at every position in the field ofview, which is applicable for the nanoparticles of this invention. Anapproximate reconstruction is possible, and a high qualityreconstruction can be obtained. Sharp, high quality projectionreconstruction ¹⁹F images may be obtained from perfluorocarbon systemsby deconvolving the spectrum from the projections prior toreconstruction (Busse, L. J., et al. Medical Physics, 13: 518-524,1986). The deconvolution is most efficiently carried out in thetime/k-space domain. A complex reference FID (if the spatialreconstruction is performed on FIDs) corresponding to the spectrum (withno gradients applied) is inverted, and then multiplied by a windowfunction to avoid the blowup at long times where the FID is small inmagnitude. The window function is constructed as a Weiner filter tomaintain an optimal adaptive approach that takes into account theinstantaneous relative magnitudes of signal and noise power in the FIDso that random noise is not unduly amplified. The method is equallyapplicable to both FID and echo based reconstructions. Because thechemical shift and J-coupling have different behavior as a function offield strength, it is essential that the reference data be obtained atthe field at which imaging is carried out. Both FID and gradient echoimaging require an FID reference function, whereas spin echo imagingrequires a spin echo reference function. The projection (or frequencyencoded) data are multiplied by the inverted/windowed reference on apixel by pixel basis to yield the deconvolved projections which may thenbe used in a conventional reconstruction.

Although most imaging today employs spin echoes, there is an advantageto using FIDs for the input to the reconstruction. Becauseperfluorocarbons exhibit significant J-coupling (which is not refocusedby a 180 degree RF pulse), in the creation of a spin echo (by means ofan RF pulse) the chemical shift and other resonance offset interactions(e.g., static magnetic susceptibility) are refocused at the echo,whereas the J-coupling is not. Therefore, significant oscillatorydephasing still occurs at the echo, and this J-modulation variesstrongly with the echo time TE. The optimum approach to eliminating thissource of artifact and signal loss is to use reconstruction fromprojections (radial imaging), such as is done for solid state MRI (Wu,Y., et al. Calcified Tissue International, 62: 512-518, 1998; Wu, Y., etal. Proceedings of the National Academy of Sciences of the United Statesof America, 96: 1574-1578, 1999; Wu, Y., et al. Magnetic Resonance inMedicine, 50: 59-68, 2003; Ramanathan, C. and Ackerman, J. L. MagneticResonance in Medicine, 41: 1214-1220, 1999). We have extensiveexperience with projection reconstruction in two and three dimensions,and can use software developed in house for this work.

Images obtained are analyzed for spatial resolution (in phantoms), forsignal to noise ratio and contrast to noise ratio. These results may becorrelated with NIRF, immunohistochemical, and histological image dataacquired from the same animals.

In vivo NIRF imaging of uMUC-1-expressing tumors in mouse models ofhuman cancer may also be performed. Tumor bearing mice injected with theprobe above are used. Near-infrared reflectance optical imaging isperformed using a whole mouse imaging system as described in (Mahmood,U., et al. Radiology, 213: 866-870, 1999). Cy5.5 fluorescence ismeasured with the appropriate filters, and mice are monitored for aperiod of 3 to 4 weeks post injection, with probe assessed as a functionof NIRF signal intensity. As the tumors grows, signal intensity isplotted as a function of tumor volume, which is obtained from calipermeasurements and an indirect calculation of tumor volume based on thedoubling time for the particular tumor. Animals are sacrificed after thecompletion of MR and NIRF imaging sessions; tumors are excised andsubjected to NIRF imaging as described.

¹²⁵I nuclear imaging may also be used, for example for imaging ofuMUC-1-expressing tumors in mouse models of human cancer. The uptakeratio and the time course of the probe accumulation may be collected,with imaging at successive time points.

Probes are prepared to inject approximately 3.7 GBq of activity in eachexperiment. Measurements may be performed using a low-energyhigh-resolution collimator on a large field of view Isocam II (IsocamTechnologies Inc., Castana, Iowa) gamma camera. Subjects are placed flaton the surface of the collimator and imaged. Subsequently, pinhole SPECTmay be performed using the same camera. Subjects are placed in avertical position in front of a pinhole collimator mounted on one of theheads of the Isocam H (Isocam Technologies Inc., Castana, Iowa) gammacamera available at the MIT Nuclear Science and Engineering Department.The subject may be positioned at a distance from the pinhole sufficientfor the acquisition of a whole body 2D image, and 2d images will becorrected for parallax. The subject is then moved axially to the heightof the lesion. Fiducial markers will be placed next to the subject, toaid the estimate of the axial shift if necessary. The support is movedcloser to the pinhole (˜3 cm radius of rotation) and rotated in a stepand shoot protocol for the acquisition of 60 to 180 angular projections.Literature methods will be used for a careful registration of theimaging parameters, notably the center of rotation. All these techniquesare well-established and proven in the nuclear imaging field. Forexamples of pinhole SPECT see (Acton, P. D., et al. European Journal ofNuclear Medicine, 29: 691-698, 2002; Schramm, N. U., et al. IEEETransactions on Nuclear Science, 50: 315-320, 2003; Moore, R. H., et al.Cancer, 32: 987, 1991; Strand, S.-E. et al. Cancer, 73: 981-984, 1994;Weber, D. A. et al. Journal of Nuclear Medicine, 35: 342-348, 1994;Jaszczak, R. J., et al. Physics in Medicine and Biology, 39: 425-437,1994; Ishizu, K. et al. Journal of Nuclear Medicine, 36: 2282-2287,1995; Booij, J., et al. European Journal of Nuclear Medicine, 29:1221-1224, 2002; Acton, P. D. and Kung, H. F. Nucl Med Biol, 30:889-895, 2003.).

Alternatives/Data analysis. In the very unlikely event that 2D and 3Dpinhole imaging should fail, parallel hole collimator images will beacquired to obtain low-resolution estimates of the biodistribution ofthe radiotracer. Given the large Fov of the camera, several animals canbe evaluated at the same time. Pinholes of different diameters (e.g.0.25, 0.5, 1 and 2 mm) will be designed and fabricated. The availabilityof several pinholes will allow the optimization of theresolution-sensitivity trade-off for image quality. The best pinholediameter will be chosen in preliminary phantom experiments with phantomsrepresentative of the expected uptake ratios. Phantom images will beevaluated for resolution and contrast recovery via region of interestanalysis. Projection data will be reconstructed with an Ordered SubsetExpectation Maximization (OSEM) iterative algorithm. Regions of interestwill be drawn in the reconstructed image to evaluate uptake ratios fromthe ratio of counts in a target and in a background region. To increasesensitivity, data can be acquired on opposite sides of the animal byusing the second head of the camera, for which a second set of pinholeswill be fabricated.

In order to follow intra-tumoral probe distribution at the microscopiclevel, immunohistochemistry and histologic evaluation of the tumors willbe correlated with results of MR, NI and NIRF studies.

Colocalization of the MR, NI and NIRF signals with staining for uMUC-1,FITC, and Cy5.5 fluorescence may be assessed. After imaging sessions,tumors are excised and snap frozen in liquid nitrogen.Immunohistochemistry probing sections with mouse monoclonal antibodiesB423 (VU4H5) against uMUC-1 60-mer tandem repeat (Biomeda, Foster City,Calif.) is followed by incubation with PE-labeled rabbit anti-mouseantibody (Pharmingen, San Diego, Calif.). Dual channel fluorescencemicroscopy is performed on consecutive sections.

Microscopic sections are digitized using, for example, a PolaroidSprintScan 35-mm scanner (Polaroid Corporation, Cambridge, Mass.) and aPathScan Enabler System (Meyer Instruments, Inc., Houston, Tex.). MR andNIRF images and corresponding digitized histological sections aredisplayed in a graphics package, such as, for example, Photoshop™ andmatching structures are identified, as described (Benveniste, H., et al.Proceedings of the National Academy of Sciences of the United States ofAmerica, 96: 14079-14084, 1999). The shape and arrangement of bloodvessels within the tissue may be used as landmarks. Approximationbetween histological sections and MR images may be further improved bymodifying the imaging plane in the volumetric MR data. The volumes ofindividual hypointense spots on the 3D MR images are measured andcorrelated to corresponding staining/fluorescence microscopy.

Example 11 Correlating In Vivo Imaging Data with Biological Function

The in vitro cytotoxicity of therapeutic probes in uMUC-1-positive celllines and normal cells may also be examined. Incubating the targetuMUC-1-positive and uMUC-1-negative tumor cells and normal cells withempty nanoparticles and nanoparticles containing the anti-cancer agent,with and without targeting peptide, may be assessed for effects on celldeath, which may be determined by various assays.

To determine cell death, methods may include incubating uMUC-1-positive,uMUC-1-negative, and normal cells with increasing concentrations andvarious nanoparticle types for time periods ranging from 6 hours to 3days at 37° C. in a humidified CO₂ atmosphere, followed by extensivewashing with media. Cell samples at each time point and for eachparticle concentration may be subjected to cell viability assays withMTT (mitochondrial function), caspase activity (apoptosis), and 7-AAD(membrane integrity). The MTT Assay (Molecular Probes, Vybrant® MTT CellProliferation Assay Kit V-13154) is a measure of the reducingenvironment in cells. The MTT reagent is a water soluble tetrazoliumcompound (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)that is reduced to an insoluble formazan product. A known concentrationof cells, for example, is resuspended in DPBS containing 20 mM glucose.20 μl of WIT reagent (5 mg/mL) and 100 ml of cell suspension will beadded to each well and the microplate will be placed in an incubator at37° C. for 4 hours. A 100 μl aliquot of a 10% (w/v) SDS and 0.01 M HClsolution is added to each well and incubated at 37° C. for 4-18 hours.The absorbance at 570 nm is determined on an ELISA plate reader andcompared with controls. Caspase activity is measured, for example, withthe Guava MultiCaspase Kit, which determines the fractions of a cellpopulation that are live, early apoptotic, late apoptotic, and dead.This assay measures caspase activity as well as membrane integrity. Theassay kit contains SR-VAD-FMK(sulforhodamine-valyl-alanyl-aspartyl-fluoromethylketone), afluorochrome-conjugated inhibitor of caspases which binds to apoptoticcells, and 7-AAD, a membrane integrity dye which stains late apoptoticand dead cells. SR-VAD-FMK penetrates all cells but only binds to activecaspases and can be washed away from non-apoptotic cells. 100 ml of cellsuspension in Apoptosis Wash Buffer will be stained with SR-VAD-FMK andincubated for 1 hour at 37° C. The cells are washed three times withApoptosis Wash Buffer, after which 5 ml of 7-AAD reagent is added toeach sample and incubated for 10 minutes at room temperature. Thesamples are analyzed using the Guava Personal Cell Analysis System(Guava Technologies), a novel flow cytometer.

Other methods may be used to assess cytotoxic effects of specificagents, for example as outlined herein, when doxorubicin and ¹³¹I areincorporated. Once internalized by the cell ¹³¹I remains attached to thepolymer to which it is covalently bound, thus ¹³¹I may be conjugated tothe polymer. Since the antitumor activity of doxorubicin requires directinteractions with DNA or DNA topoisomerase, doxorubicin should bereleased from polymer, in order to gain access to the nucleus of thecell. Nanoparticles/micelles loaded with radiolabel and doxorubicin maybe diluted in PBS (1:1 by volume), loaded in a dialysis cassette(molecular weight cutoff of 10,000 Da), and dialyzed against 50% PBS at37° C. At different time points, aliquots may be measured fordoxorubicin concentration. The results will be expressed as t_(1/2)(time in which 50% of drug exits the nanoparticle/micelle).

Pharmacokinetic data (biodistribution, blood half-life) in terms of drugaffinity and accumulation in uMUC-1-positive primary tumors andmetastasis in vivo may be assessed.

In vivo cytotoxicity studies in for example, a mouse model of humancancer may be conducted as well. Survival time and tumor volumefollowing administration of targeted therapeutic probes containingdoxorubicin may be evaluated. For example, nu/nu mice injectedsubcutaneously with uMUC-1-positive and uMUC-1-negative tumor cellsuspensions are assessed for tumor volume via calipers (0.5 cm indiameter). Single- and multiple-dose treatment studies may be conductedwith the drug, given intravenously. Tumor growth may be assessed twice aweek by caliper measurements. Tumor volume may be calculated using theequation for the volume of a prolate ellipsoid: (a×b²)×π/6, where a isthe larger and b is the smaller dimension of the tumor. The results maybe expressed as relative tumor volume, Vt/Vo, where Vo is the tumorvolume at the start of the treatment and Vt is the tumor volume at anygiven time point.

Results from in vivo cytotoxicity experiments may be correlated with invivo ¹⁹F MR, NIRF, and/or nuclear imaging. The time course ofnanoparticle/micelle accumulation by imaging during treatment may beevaluated. In addition, histology of excised tumors may be correlatedwith apoptosis and/or differential uMUC-1 expression in vivo in responseto therapy. For the latter, quantitative RT-PCR may be used to determinechanges in uMUC-1 expression. The following primers and probes specificfor the MUC-1 5′ non-repeat region may be used:

Forward primer,  (SEQ ID NO: 3) 5′-ACAGGTTCTGGTCATGCAAGC-3′;Reverse primer,  (SEQ ID NO: 4) 5′-CTCACAGCATTCTTCTCAGTAGAGCT-3'TaqMan Probe,  (SEQ ID NO: 5)5′-FAM-TGGAGAAAAGGAGACTTCGGCTACCCAGA-TAMRA-3′.

Although the invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives, modificationsand variations will be apparent to those skilled in the art, any ofwhich are to be considered as part of this invention.

1-166. (canceled)
 167. A method of imaging a cell, the method comprisingcontacting a cell with an amphiphilic polymer characterized by thestructure of the general formula I:

wherein R is a hydroxyl, O-alkyl, O-Acyl, O-Activating group, SH,S-alkyl, or an acid activating group including halogen, O-vinyl,O-allyl, O-aryl, OCOalkyl, OCOaryl, OCH₂CF₃, NH₂, a fluorochrome, anindole-containing compound, an antibody or antibody fragment, a peptide,an oligonucleotide, a drug, a ligand for a biological target, animmunoconjugate, a chemomimetic functional group, a glycolipid, alabelling agent, an enzyme, a metal ion chelate, an enzyme cofactor, acytotoxic compound, a growth factor, a hormone, a cytokine, a toxin, aprodrug, an antimetabolite, a microtubule inhibitor, a radioactivematerial, a targeting moiety; R′ is OH, NH₂, SH; each R₁ group is,independently, H,

a fluorochrome, an indole-containing compound, an antibody or antibodyfragment, a peptide, an oligonucleotide, a drug, a ligand for abiological target, an immunoconjugate, a chemomimetic functional group,a glycolipid, a labelling agent, an enzyme, a metal ion chelate, anenzyme cofactor, a cytotoxic compound, a growth factor, a hormone, acytokine, a toxin, a prodrug, an antimetabolite, a microtubuleinhibitor, a radioactive material, a perfluorocarbon, aperfluorocarbon-R₄, a perfluorocarbon-OR₄,

each R₂ group is, independently, a fluorochrome, an indole-containingcompound, an antibody or antibody fragment, a peptide, anoligonucleotide, a drug, a ligand for a biological target, animmunoconjugate, a chemomimetic functional group, a glycolipid, alabelling agent, an enzyme, a metal ion chelate, an enzyme cofactor, acytotoxic compound, a growth factor, a hormone, a cytokine, a toxin, aprodrug, an antimetabolite, a microtubule inhibitor, a radioactivematerial, a perfluorocarbon, a perfluorocarbon-R₄, aperfluorocarbon-OR₄,

each R₃ group is, independently,

a hydrogen, a hydroxyl, O-alkyl, SH, S-alkyl, or an acid activatinggroup including halogen, O-vinyl, O-allyl, O-aryl, OCOalkyl, OCOaryl,OCH₂CF₃, NH₂, a fluorochrome, an indole-containing compound, an antibodyor antibody fragment, a peptide, an oligonucleotide, a drug, a ligandfor a biological target, an immunoconjugate, a chemomimetic functionalgroup, a glycolipid, a labelling agent, an enzyme, a metal ion chelate,an enzyme cofactor, a cytotoxic compound, a growth factor, a hormone, acytokine, a toxin, a prodrug, an antimetabolite, a microtubuleinhibitor, a radioactive material, a targeting moiety; each R₄ group is,independently, an alkyl group, an alkylene group, a carboxylate group, acarboxylic acid group, an amino group, an ammonium group, an alkoxylgroup, a hydroxyl group or another nitrogen, oxygen or sulfur-containinggroup; each A group is, independently, O, NH, S, a fluorochrome,

an indole-containing compound, an antibody or antibody fragment, apeptide, an oligonucleotide, a drug, a ligand for a biological target,an immunoconjugate, a chemomimetic functional group, a glycolipid, alabeling agent, an enzyme, a metal ion chelate, an enzyme cofactor, acytotoxic compound, a growth factor, a hormone, a cytokine, a toxin, aprodrug, an antimetabolite, a microtubule inhibitor, a radioactivematerial, a targeting moiety, an acyl group, an aryl group, a linear orbranched alkenyl group, a linear or branched alkyl group, wherein saidalkyl, alkenyl or aryl group is substituted with a perfluorocarbon,perfluorocarbon-R₄, perfluorocarbon-OR₄, perfluorocarbon-OR₄, or

n, m, p, p′ and x are integers; and q is an integer between 0-10,wherein the amphiphilic polymer further comprises a targeting agent, themethod further comprising imaging said cell, whereby said polymerenables the imaging of said cell.